Polymeric alpha-hydroxy aldehyde and ketone reagents and conjugation method

ABSTRACT

Provided herein are polymeric α-hydroxy aldehyde or α-hydroxy ketone reagents which can be conjugated to amine-containing compounds to form stable conjugates in a single-step reaction. In selected embodiments, the polymeric reagent itself incorporates an internal proton-abstracting (basic) functional group, to promote more efficient reaction. The substituent is appropriately situated, via a linker if necessary, to position the group for proton abstraction, preferably providing a 4- or 5-bond spacing between the abstracting atom and the hydrogen atom on the α-carbon. Also provided are methods of using the reagents and stable, solubilized conjugates of the reagents with biologically active compounds. In preferred embodiments, the polymeric component of the reagent or conjugate is a polyethylene glycol.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/937,378, filed Jul. 23, 2020, now U.S. Pat. No. 11,220,575, which isa divisional of U.S. patent application Ser. No. 15/867,524, filed Jan.10, 2018, now U.S. Pat. No. 10,759,906, which is a continuation of U.S.patent application Ser. No. 15/408,037, filed Jan. 17, 2017, now U.S.Pat. No. 9,908,970, which is a continuation of U.S. patent applicationSer. No. 14/951,035, filed Nov. 24, 2015, now U.S. Pat. No. 9,579,392,which is a divisional of U.S. patent application Ser. No. 13/925,074,filed Jun. 24, 2013, now U.S. Pat. No. 9,228,053, which is acontinuation of U.S. patent application Ser. No. 13/063,720, filed May16, 2011, now U.S. Pat. No. 8,492,503, which is a 35 U.S.C. § 371application of International Application No. PCT/US2009/005092, filedSep. 11, 2009, designating the United States, which claims the benefitof priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 61/096,112, filed Sep. 11, 2008, all of which areincorporated by reference herewith in their entireties.

REFERENCES

-   Crestia, D. et al., Tetrahedron: Asymmetry 12:869-876 (2001).-   Hermanson, G. T. Bioconjugate Techniques, Academic Press, San Diego,    1996, pp. 37-38.-   Hodge, J. E., Advan. Carbohyd. Chem. 10:169 (1955).-   Isbell, H. S. et al., J. Org. Chem. 23:1309 (1958).-   Lemieux, R. U., in P. de Mayo, Molecular Rearrangements, Pt. 2, p.    753 (1964).-   Micheel et al., Ann. 658:120 (1962).-   Wrodnigg, T. M and Eder, B., Topics in Current Chemistry 215:115-152    (2001).

BACKGROUND OF THE INVENTION

In recent years, human therapeutics have expanded past traditional smallmolecule drugs and into the realm of biopharmaceuticals. The discoveryof novel proteins and peptides has led to the development of numerousprotein and polypeptide biopharmaceuticals. Unfortunately, proteins andpolypeptides, when utilized as therapeutics, often exhibit propertiesthat make them extremely difficult to formulate or administer, such asshort circulating half lives, immunogenicity, proteolytic degradation,and low solubility. One approach for improving the pharmacokinetic orpharmacodynamic properties of biopharmaceuticals is the conjugation tonatural or synthetic polymers, such as polyethylene glycol (PEG). Thecovalent attachment of PEG to a therapeutic protein can provide a numberof advantages, such as (i) shielding antigenic epitopes of the protein,thus reducing its reticuloendothelial clearance and recognition by theimmune system, (ii) reducing degradation by proteolytic enzymes, and(iii) reducing renal filtration.

Much effort has been spent on the development of polymer derivatives forcoupling to biopharmaceuticals such as peptides, and in particular, onthe development of polymer derivatives for coupling to reactive aminogroups of proteins. Examples of such PEG derivatives include PEGdichlorotriazine, PEG tresylate, PEG succinimidyl carbonate, PEGcarbonylimidazole, and PEG succinimidyl succinate. However, thesereagents can suffer from drawbacks such: undesirable side reactionsunder the reaction conditions necessary to effect coupling, lack ofselectivity, and/or the formation of weak (i.e., unstable) linkagesbetween the biopharmaceutical and the PEG.

Aldehyde-terminated PEGs, such as PEG propionaldehyde and PEGacetaldehyde (see, for Example, U.S. Pat. Nos. 5,252,714 and 5,990,237,respectively) provide the advantage of selectivity in their attachmentchemistry. However, when a polymeric aldehyde or ketone is used forconjugation with a polypeptide or similar molecule having a reactiveamine group, it is necessary to include a reduction step to stabilizethe imine linkage that forms during the conjugation step.

SUMMARY

In one aspect, the invention provides a polymeric reagent having thestructure I.

where

R¹ is selected from H, lower alkyl, and alkoxyalkyl, and is preferably Hor methyl;

R⁴ is a two- or three-carbon chain which may be substituted with one ormore groups selected from alkyl, alkenyl, aryl, alkoxy, halo, cyano, anda water soluble polymer, wherein the carbon adjacent to Cα is notsubstituted with hydroxy, and wherein two substituents on R⁴ maytogether form an aliphatic or aromatic ring; and

NR⁵ is a secondary or tertiary amino group which is linked to a watersoluble polymer POLY, preferably a polyethylene glycol, via an optionalspacer group Z, where R⁵ is hydrogen or an alkyl group, which may form aring with spacer group Z.

In one embodiment, R¹ is H, such that the reagent contains an α-hydroxyaldehyde.

In further embodiments, R⁴ in structure I is unsubstituted or issubstituted with lower alkyl. R⁴ in structure I is preferably athree-carbon chain, and may be saturated or unsaturated. Moreparticularly, reagents in which R⁴ is a saturated three-carbon chain mayhave the structure Ia:

where R¹, NR⁵, Z and POLY are as defined above, each of the substituentsR^(c), R^(c′), R^(d), and R^(d′) is independently selected fromhydrogen, alkyl, alkenyl, aryl, alkoxy, halo, cyano, hydroxy, and awater soluble polymer, and each of the substituents R^(b) and R^(b′) isindependently selected from hydrogen, alkyl, alkenyl, aryl, alkoxy,halo, cyano, and a water soluble polymer, wherein at most one of thesesubstituents is a water soluble polymer, and wherein any two of thesesubstituents can together form an aliphatic ring.

In selected embodiments of this structure, the substituents R^(b),R^(b′), R^(c), R^(c′), R^(d), and R^(d′) are independently selected fromhydrogen and alkyl, wherein any two such alkyl substituents, preferablyon adjacent carbon atoms, can together form a 5- to 7-membered aliphaticring.

In one embodiment, the group R⁵ is methyl. Reagents of this classinclude those designated (5) and (10) herein(5-(mPEG-methyl-amino)-2-hydroxypentanal). In these exemplary reagents,each of R^(b), R^(b′), R^(c), R^(c′), R^(d), and R^(d′) in structure Iais hydrogen.

The spacer group Z, when present, preferably consists of bonds selectedfrom alkylene, ether, thioether, amide, and amine. In one embodiment,NR⁵ together with Z forms a ring to which POLY is linked, e.g. apiperazine ring to which POLY is linked via a ring nitrogen atom.Reagents of this class include those designated (11) and (12) herein(5-mPEG-piperazine-2-hydroxy-pentanal). In these exemplary reagents,each of R^(b), R^(b′), R^(c), R^(c′), R^(d), and R^(d′) in structure Iais hydrogen.

In another aspect, the invention provides a polymeric reagent having thestructure II:

where

POLY is a water soluble polymer, preferably a polyethylene glycol, whichmay include a linker moiety, as described herein, linking it to(CR²R³)_(m);

R¹ is selected from H, alkyl, hydroxyalkyl, and alkoxyalkyl, preferablyH or methyl, and more preferably H;

m is 0-12, preferably 0 to 6; and

each R² and R³ is independently selected from H, alkyl, alkylene,hydroxy, amino, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxyalkylene,aminoalkyl, iminoalkyl, carboxylic acid, alkylcarboxylic acid,phosphate, alkylphosphate, and a further water soluble polymer, whereinat most one R² or R³ group is a water soluble polymer, and wherein twosubstituents R² and R³ in (CR²R³)_(m) can together form a ring;

with the proviso that no R² or R³ on the carbon adjacent to Cα is ahydroxyl group or a 1,2,3-trihydroxypropyl group.

Preferably, at most one group R² or R³ is selected from aminoalkyl,iminoalkyl, carboxylic acid, alkylcarboxylic acid, phosphate, andalkylphosphate. Aminoalkyl and iminoalkyl can include (cyclicamino)alkyl or (cyclic imino)alkyl; i.e. where the amine nitrogen isincluded in a ring, which may itself form part of the —(CR²R³)_(m)—chain.

In further preferred embodiments, one group R² or R³ is selected fromhydroxy, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxyalkylene, amino,aminoalkyl, iminoalkyl, carboxylic acid, alkylcarboxylic acid,phosphate, and alkylphosphate; and preferably from amino, aminoalkyl,iminoalkyl, carboxylic acid, and alkylcarboxylic acid; and this group(R² or R³) and the value of m are selected such that an oxygen ornitrogen atom on R² or R³ is separated from Cα by a four-bond path.

In one embodiment, POLY is mPEG-O—, m is 3, and —(CR²R³)_(m)— isselected from —(CH₂)₃— and —CH₂—CH(OH)—CH₂—. The compound designated 4herein (5-mPEG-2-hydroxypentanal) is an example of such a structurewhere —(CR²R³)_(m)— is —(CH₂)₃—.

Reagents in which —(CR²R³)_(m)— include the moiety —CH₂—CH(OH)—CH₂—attached to Cα are based on 3-deoxy analogs of reducing sugars, such as3-deoxy ribose. One example is the compoundmPEG-O—CH₂—CH(OH)—CH₂—CH(OH)—C(O)H, designated herein as 13. Otherpossible reagents include those derived from the 3-deoxy analogs of, forexample, xylose, arabinose, glucose, galactose, or mannose. Five-carbonsugars such as ribose, xylose, and arabinose are preferred. Suchreagents are advantageous over the corresponding derivatives of thenative sugars, because the absence of the 3-hydroxyl group in theeventual conjugated product reduces opportunities for degradation of theconjugated product.

In another embodiment, POLY is mPEG-O—, m is 1, R¹ is H, R² is H, and R³is iminoalkyl. The compound designated 3 herein is an example of such astructure, where R³ is (4-imidazolyl)methyl.

In still further embodiments, —(CR²R³)_(m)— in structure II is definedby —CH₂—CHR^(x)—C(NH₂)R^(y)—, where R^(x) and R^(y) form a 5- to7-membered aliphatic ring to which POLY is linked. Such reagents includethose illustrated as follows, where n is 0 to 2:

The amino group may be replaced with methylamino or dimethylamino.Reagents similar to those illustrated above but including an aromaticring are also provided, as described further below.

As noted above, a linker moiety may be used to connect the polymercomponent POLY to the functional portion of the reagent of structure II,or to connect polymer segments making up the component POLY. The natureof the linker, when present, is described further below. Typically, alinker includes a functional group such as an amide, an ester, aurethane, or a urea, optionally containing methylene or other alkylenegroups flanking either side of the single functional group.Alternatively, a linker can be an alkylene chain, optionally containingone or more oxygen or sulfur atoms (i.e., an ether or thioether).

In a related aspect, the invention provides a polymeric α-hydroxyaldehyde or ketone reagent having the structure III:

where

POLY is a water soluble polymer, such as a polyethylene glycol, whichmay include a linker moiety, as described herein, linking it to thearomatic ring; and

(i) R⁷ is —CH(OH)—C(═O)—R¹, and R⁸ is selected from —C(═O)OH, —CH₂OH,—C(═O)N(R¹)₂, —CH₂N(R¹)₂, —OH, and —N(R¹)₂, and is preferably selectedfrom —C(═O)OH, —CH₂OH, —C(═O)N(R¹)₂, and —CH₂N(R¹)₂; or

(ii) R⁷ is —CHR¹CH(OH)—C(═O)—R¹, and R¹ is —OH or —N(R¹)₂;

where each R¹ is independently selected from H, lower alkyl, andalkoxyalkyl, and is preferably independently selected from H and methyl.In selected embodiments, R¹ attached to C(═O) is H, such that thereagent contains an α-hydroxy aldehyde. In other embodiments, each R¹ isH.

Exemplary reagents of this class include the compounds designated hereinas 6, where R⁷ is —CH(OH)—C(═O)—H and R⁸ is —C(═O)OH; 7, where R⁷ is—CH(OH)—C(═O)—H and R⁸ is —OH; and 8, where R⁷ is —CH₂CH(OH)—C(═O)—H andR⁸ is —OH. Analogous reagents include those in which the phenolichydroxy group (OH) is replaced by NH₂ or N(CH₃)₂ (that is, R⁸ is —NH₂ or—N(CH₃)₂).

In another aspect, the invention provides a method of conjugating awater soluble polymer with a compound B—NH₂ having a reactive aminegroup, the method comprising contacting the compound B—NH₂ with apolymeric reagent comprising a water soluble polymer and an α-hydroxyaldehyde or α-hydroxy ketone end group, respectively, thereby forming aconjugate in which the water soluble polymer is linked to a moietyhaving the structure —C(═O)—CHR¹—NH—B, where R¹ is hydrogen or methyl,respectively, and NH—B represents the residue of the amine-containingcompound. Such contacting is carried out in a suitable aqueous ororganic solvent, under conditions as needed for reaction, includingcatalysis if necessary.

In preferred embodiments, the polymeric reagent further comprises aproton-abstracting functional group containing a proton-abstracting atomselected from oxygen and nitrogen, situated such there is a 3- or 4-bondspacing, preferably a 4-bond spacing, between the proton-abstractingatom and the α-carbon of the α-hydroxy aldehyde or α-hydroxy ketone.Such reagents are able to undergo a “self-catalyzed” conjugationreaction, as described further below.

In selected embodiments, the method employs a polymeric reagent ofstructure I/Ia, II, or III as described herein.

Additional preferred embodiments of the method generally employpreferred embodiments of the reagents I/Ia, II and III as describedherein. The amine-containing compound is typically a biologically activeor biologically relevant compound, such as a polypeptide or protein.

In a related aspect, the invention provides conjugates which can beformed by such a method. For example, the invention provides a polymericconjugate having the structure IV:

where

R¹ is selected from H, lower alkyl, and alkoxyalkyl, and is preferably Hor methyl;

R⁴ is a two- or three-carbon chain which may be substituted with one ormore groups selected from alkyl, alkenyl, aryl, alkoxy, halo, cyano, anda water soluble polymer,

wherein the carbon adjacent to the carbonyl carbon is not substitutedwith hydroxy, and wherein two substituents on R⁴ may together form analiphatic or aromatic ring; and

NR⁵ is a secondary or tertiary amino group which is linked to a watersoluble polymer POLY, such as a polyethylene glycol, via an optionalspacer group Z, where R⁵ is hydrogen or an alkyl group, which may form aring with spacer group Z; and

—NH—B represents the residue of an amine-containing biologically activecompound.

Selected embodiments of the conjugate include those which correspond toselected embodiments of reagent I/Ia described herein.

Further conjugates of the invention include those of structure V:

wherein:

POLY is a water soluble polymer, such as a PEG, which may include alinker moiety, as described herein, linking it to (CR²R³)_(m);

R¹ is selected from H, alkyl, hydroxyalkyl, and alkoxyalkyl;

m is 0-12; and

each R² and R³ is independently selected from H, alkyl, alkylene,hydroxy, amino, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxyalkylene,aminoalkyl, iminoalkyl, carboxylic acid, alkylcarboxylic acid,phosphate, alkylphosphate, and a further water soluble polymer, whereinat most one R² or R³ group is a water soluble polymer, and wherein twosubstituents R² and R³ in (CR²R³)_(m) can together form a ring,preferably an aliphatic ring; and

—NH—B represents the residue of an amine-containing biologically activecompound, such as a polypeptide or protein.

Preferably, no R² or R³ on the carbon adjacent to the carbonyl carbon isa hydroxyl group or a 1,2,3-trihydroxypropyl group.

Additional preferred embodiments of the conjugate generally correspondto preferred embodiments of the reagent II as described herein.

Further conjugates of the invention include those of structure VI:

where

(i) R⁷ is C(═O)—CHR¹—NH—B, and R⁸ is selected from —C(═O)OH, —CH₂OH,—C(═O)N(R¹)₂, —CH₂N(R¹)₂, —OH, and —N(R¹)₂, and is preferably selectedfrom —C(═O)OH, —CH₂OH, —C(═O)N(R¹)₂, and —CH₂N(R¹)₂; or

(ii) R⁷ is —CHR¹—C(═O)—CHR¹—NH—B, and R¹ is —OH or —N(R¹)₂; POLY is awater soluble polymer, such as a PEG, which may include a linker moiety,as described herein, linking it to the aromatic ring; and

—NH—B represents the residue of an amine-containing biologically activecompound;

where each R¹ is independently selected from H, lower alkyl, andalkoxyalkyl, and preferably from H and methyl.

Additional preferred embodiments of the conjugate generally correspondto preferred embodiments of the reagent III as described herein, wherethe groups R⁷ and R⁸ are defined accordingly.

In the conjugates of structures IV, V, and VI, the amine-containingcompound is typically a biologically active or biologically relevantcompound, such as a polypeptide or protein.

These and other aspects of the invention will become apparent uponreview of the following description and accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an SDS-PAGE gel obtained from electrophoresis of reactionmixtures of conjugation reactions of a protein, lysozyme (1 mg/mL), withthe polymeric reagents mPEG₁₀₀₀₀-HP-ALD (invention compound 4) andmPEG₁₀₀₀₀-butyrALD (prior art compound), run under different reactionconditions, as described in Example 6a.

Lane:

1) Benchmark ladder,

2) lysozyme (Lyz),

3) Lyz+mPEG_(10K)-HP-ALD (4), pH 6.5,

4) Lyz+mPEG_(10K)-HP-ALD (4), pH 7.5,

5) Lyz+mPEG_(10K)-HP-ALD (4), pH 9.0,

6) Lyz+mPEG_(10K)-HP-ALD (4), pH 7.5+NaCNBH₃,

7) Lyz+mPEG_(10K)-butyrALD (20 kD), pH 7.5,

8) Lyz+mPEG_(10K)-butyrALD (20 kD), pH 7.5+NaCNBH₃.

FIG. 2 shows an SDS-PAGE gel obtained from electrophoresis of reactionmixtures of conjugation reactions of Lysozyme (1 mg/mL) and thepolymeric reagents mPEG_(10K)-HP-acetal (acetal of invention compound4), mPEG_(10K)-MAHP-ALD (invention compound 5, “self-catalyzing”) andits acetal, run under different reaction conditions, as described inExample 6b.

Lane:

1) Benchmark ladder,

2) lysozyme (Lyz),

3) Lyz+mPEG_(10K)-HP (4) acetal, pH 6.5,

4) Lyz+mPEG_(10K)-HP (4) acetal, pH 9.0,

5) Lyz+mPEG_(10K)-MAHP (5) acetal, pH 6.5,

6) Lyz+mPEG_(10K)-MAHP (5) acetal, pH 9.0,

7) Lyz+mPEG_(10K)-MAHP-ALD (5), pH 6.5,

8) Lyz+mPEG_(10K)-MAHP-ALD (5), pH 9.0,

9) Lyz+mPEG_(10K)-MAHP (5) acetal (purified, 95% sub), pH 6.5,

10) Lyz+mPEG_(10K)-MAHP (5) acetal (purified, 95% sub), pH 9.0.

FIG. 3 shows an SDS-PAGE gel obtained from electrophoresis of reactionmixtures of conjugation reactions of a protein, lysozyme (1 mg/mL), withthe polymeric reagents mPEG_(10K)-HP-ALD (invention compound 4),mPEG_(10K)-MAHP-ALD (invention compound 5, “self-catalyzing”) and itsacetal, and mPEG_(10K)-butyrALD (prior art compound), run underdifferent reaction conditions, as described in Example 6c.

Lane:

1) Lyz+mPEG_(10K)-HP-ALD (4), pH 6.5,

2) Lyz+mPEG_(10K)-HP-ALD (4), pH 7.5,

3) Lyz+mPEG_(10K)-MAHP-ALD (5) (purified, 95% sub), pH 6.5,

4) Lyz+mPEG_(10K)-MAHP-ALD (5) (purified, 95% sub), pH 7.5,

5) Lyz+mPEG_(10K)-MAHP (5) acetal (purified, 95% sub), pH 7.5,

6) Lyz+mPEG_(20K)-butyrALD, pH 7.5,

7) Lyz+mPEG_(20K)-butyrALD, pH 6.5+NaCNBH₃,

8) Lyz+mPEG_(20K)-butyrALD, pH 7.5+NaCNBH₃

9) lysozyme,

10) Benchmark ladder.

FIG. 4 shows an SDS-PAGE gel obtained from electrophoresis of reactionmixtures of conjugation reactions of a protein, lysozyme (1 mg/mL), withthe polymeric reagents mPEG_(20K)-MAHP-ALD (invention compound 10,“self-catalyzing”) and mPEG_(10K)-Pip-HP-ALD (invention compound 11,“self-catalyzing”), run under different reaction conditions, asdescribed in Example 11.

Lane:

1) Benchmark ladder,

2) Lysozyme (Lyz),

3) Lyz+mPEG_(10K)-Pip-HP-ALD (11), 25×, pH 6.5,

4) Lyz+mPEG_(10K)-Pip-HP-ALD (11), 50×, pH 6.5,

5) Lyz+mPEG_(10K)-Pip-HP-ALD (11), 50×, pH 9.1,

6) Lyz+mPEG_(10K)-Pip-HP-ALD (11), 25×, pH 6.0,

7) Lyz+mPEG_(10K)-Pip-HP-ALD (11), 50×, pH 6.0,

8) Lyz+mPEG_(20K)-MAHP-ALD (10), 25×, pH 6.5,

9) Lyz+mPEG_(20K)-MAHP-ALD (10), 50×, pH 6.5,

10) Lyz+mPEG_(20K)-MAHP-ALD (10), 50×, pH 9.1.

FIGS. 5A-B show SDS-PAGE gels obtained from electrophoresis of reactionmixtures of conjugation reactions of a 55 kDa protein (1 mg/mL) with thepolymeric reagents mPEG_(20K)-HP-ALD (invention compound 9) andmPEG_(20K)-MAHP-ALD (invention compound 10, “self-catalyzing”), rununder different reaction conditions, as described in Example 12. FIG.5A, Barium/Iodine stain; FIG. 5B, Coomassie stain.

Lane:

1) MW markers,

2) 1 μg mPEG_(20K)-HP-ALD (9),

3) 3 μg unmodified 55 kDa protein,

4) mPEG_(20K)-HP-ALD (9)/55 kDa protein; pH 6.5,

5) mPEG_(20K)-HP-ALD (9)/55 kDa protein; pH 7.5,

6) mPEG_(20K)-HP-ALD (9)/55 kDa protein; pH 9.0,

7) 1 μg mPEG_(20K)-HP-MALD (10),

8) 3 μg unmodified 55 kDa protein,

9) mPEG_(20K)-HP-MALD (10)/55 kDa protein; pH 6.5,

10) mPEG_(20K)-HP-MALD (10)/55 kDa protein; pH 7.5,

11) mPEG_(20K)-HP-MALD (10)/55 kDa protein; pH 9.0,

12) Blank.

FIGS. 6A-B show SDS-PAGE gels obtained from electrophoresis of reactionmixtures of conjugation reactions of lysozyme (1 mg/mL) with theindicated polymeric reagents (50×) at pH 6.5 and 9.0 in 0.2 M phosphatebuffer, as described in Example 14. FIG. 6A: reactions at r.t. for 1day; FIG. 6B, reactions at 5° C. for 1 day.

Lane:

1) Lyz+mPEG_(20K)-HP-ALD (9), pH 6.5,

2) Lyz+mPEG_(20K)-HP-ALD (9), pH 9.0,

3) Lyz+mPEG_(20K)-MAHP (10), pH 6.5,

4) Lyz+mPEG_(20K)-MAHP (10), pH 9.0,

5) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 6.5,

6) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 9.0,

7) Lyz+mPEG_(20K)-butryALD, pH 6.5,

8) Lyz+mPEG_(20K)-butryALD+NaCNBH₃, pH 6.5.

9) lysozyme (Lyz),

10) Benchmark ladder.

FIG. 6C shows SDS-PAGE gels obtained from electrophoresis of reactionmixtures of conjugation reactions of lysozyme (1 mg/mL) with theindicated polymeric reagents (50×) at pH 7.5, as also described inExample 14.

Lane:

1) Lyz+mPEG_(20K)-HP-ALD (9), 0.2 M phosphate buffer, pH 7.5,

2) Lyz+mPEG_(20K)-HP-ALD (9), 0.2 M HEPES buffer, pH 7.5,

3) Lyz+mPEG_(20K)-MAHP (10), 0.2 M phosphate buffer, pH 7.5,

4) Lyz+mPEG_(20K)-MAHP (10), 0.2 M HEPES buffer, pH 7.5,

5) Lyz+mPEG_(20K)-Pip-HP-ALD (12), 0.2 M phosphate buffer, pH 7.5,

6) Lyz+mPEG_(20K)-Pip-HP-ALD (12), 0.2 M HEPES buffer, pH 7.5,

7) Lyz+mPEG_(20K)-butryALD, 0.2 M phosphate buffer, pH 7.5,

8) Lyz+mPEG_(20K)-butryALD, +NaCNBH₃, 0.2 M phosphate buffer, pH 7.5,

9) lysozyme,

10) Benchmark ladder.

FIG. 7 shows SDS-PAGE gels obtained from electrophoresis of purifiedreaction mixtures of conjugation reactions of lysozyme (1 mg/mL) withmPEG-aldehyde 20K reagent (50×) at pH 6.5, 7.5, and 9.0, as described inExample 17.

Lane:

0) Invitrogen Mark12′

1) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 6.5;

2) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 7.5;

3) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 9.0.

FIG. 8 shows a synthetic scheme for preparation of a branched-PEGreagent of the invention, 5-ruPEG2_(20K)-piperazine-2-hydroxy-pentanal(or ruPEG2_(20K)-Pip-HP-ALD) (14).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular polymers,synthetic techniques, active agents, and the like, as such may vary.

As used in this specification and in the claims, the singular forms “a”,“an”, and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymer” includesa single polymer as well as two or more of the same or differentpolymers, reference to “a conjugate” refers to a single conjugate aswell as two or more of the same or different conjugates, reference to“an excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

“Optional” and “optionally” mean that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG”, “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are meant to encompass any water-soluble poly(ethylene oxide).Typically, PEGs for use in accordance with the invention comprise thefollowing structure “—O(CH₂CH₂O)_(m)—” where (m) is 2 to 4000. As usedherein, PEG also includes “—(CH₂CH₂O)_(m)—” and“—CH₂CH₂—O(CH₂CH₂O)_(m)—CH₂CH₂—”, depending upon whether or not theterminal oxygens have been displaced. When the PEG further comprises aspacer moiety (to be described in greater detail below), the atomscomprising the spacer moiety, when covalently attached to awater-soluble polymer segment, do not result in the formation of anoxygen-oxygen bond (i.e., an “—O—O—” or peroxide linkage). Throughoutthe specification and claims, it should be remembered that the term“PEG” includes structures having various terminal or “end capping”groups and so forth. The term “PEG” also means a polymer that contains amajority, that is to say, greater than 50%, of —CH₂CH₂O— monomericsubunits. With respect to specific forms, the PEG can take any number ofa variety of molecular weights, as well as structures or geometries suchas “branched,” “linear,” “forked,” “multifunctional,” “dendrimeric”, andthe like, to be described in greater detail below.

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled to can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like. Suitabledetectors include photometers, films, spectrometers, and the like.

“Non-naturally occurring”, with respect to a polymer or water-solublepolymer, indicates that the polymer in its entirety is not found innature. A non-naturally occurring polymer or water-soluble polymer may,however, contain one or more subunits or portions of a subunit that arenaturally occurring, so long as the overall polymer structure is notfound in nature.

A “water-soluble polymer” is any polymer that is soluble in water atroom temperature. Typically, a water-soluble polymer will transmit atleast about 75%, more preferably at least about 95% of light,transmitted by the same solution after filtering. On a weight basis, awater-soluble polymer will preferably be at least about 35% (by weight)soluble in water, more preferably at least about 50% (by weight) solublein water, still more preferably about 70% (by weight) soluble in water,and still more preferably about 85% (by weight) soluble in water. It isstill more preferred, however, that the water-soluble polymer is about95% (by weight) soluble in water and most preferred that thewater-soluble polymer is completely soluble in water.

“Molecular weight”, in the context of a water-soluble polymer of theinvention, such as PEG, can be expressed as either a number averagemolecular weight or a weight average molecular weight. Unless otherwiseindicated, all references to molecular weight herein refer to the weightaverage molecular weight. Both molecular weight determinations, numberaverage and weight average, can be made using gel permeationchromatography or other liquid chromatography techniques. Other methodsfor measuring molecular weight can also be used, such as end-groupanalysis or colligative properties (e.g., freezing-point depression,boiling-point elevation, or osmotic pressure) to determine numberaverage molecular weight, or light scattering techniques,ultracentrifugation or viscometry to determine weight average molecularweight. The polymers of the invention are typically polydisperse (i.e.,number average molecular weight and weight average molecular weight ofthe polymers are not equal), possessing low polydispersity values ofpreferably less than about 1.2, more preferably less than about 1.15,still more preferably less than about 1.10, yet still more preferablyless than about 1.05, and most preferably less than about 1.03.

An “organic radical” as used includes, for example, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryland substituted aryl.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includeethyl, propyl, butyl, pentyl, isooctyl, decyl, 3-ethyl-3-pentyl,2-methyl-1-hexyl, and the like. As used herein, “alkyl” includescycloalkyl, when three or more carbon atoms are referenced, and loweralkyl. “Alkylene” refers to an unsaturated bivalent radical (e.g.—(CH₂)_(n))—.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, preferably 1 to 4 carbon atoms, and may be straight chain orbranched, as exemplified by methyl, ethyl, n-butyl, iso-butyl, andtert-butyl. When a group is defined as “alkyl” herein, lower alkyl isgenerally a preferred embodiment.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, perfluorobutyl, etc.), preferably C₁-C₇ alkyl, more preferablyC₁-C₇ alkyl. “Alkoxyalkyl” refers to an —R—O—R group, where R is asdefined above, and is preferably unsubstituted C₁-C₇ alkyl.

“Aminoalkyl” refers to an —NHR or —NR₂ group, where R is alkyl asdefined above, and is preferably unsubstituted C₁-C₇ alkyl, and the twoR groups in —NR₂ may be the same or different. The two R groups may alsoform a five- to seven-membered ring.

“Iminoalkyl(ene)” refers to an —R′—N═R″ group, where R″ represents CH₂,CHR, or CR₂, where each R is alkyl as defined above, and the two Rgroups in —CR₂ may be the same or different. R′ is alkyl as definedabove, i.e. an sp² hybridized carbon, or alkylene, i.e. an sp²hybridized carbon forming one member of a double bond. An R in CHR orCR₂ taken together with the R′ may form a five- to seven-membered ring.As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 2 to 15 atoms in length, containing at least one double bond,such as ethenyl, n-propenyl, isopropenyl, n-butenyl, isobutenyl,octenyl, decenyl, tetradecenyl, and the like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond, ethynyl, n-butynyl, isopentynyl, octynyl, decynyl, and soforth.

“Aliphatic” refers to a group containing carbon and hydrogen which isnot aromatic. As used herein, it can refer to linear, branched, orcyclic groups. It can refer to saturated or unsaturated groups, withsaturated groups generally being preferred.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl, or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl. An aromatic moiety(e.g., Ar¹, Ar², and so forth), means a structure containing aryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule. Such groups include: lower alkyl, loweralkoxy, C₃-C₈ cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like;halo, e.g., fluoro, chloro, bromo, and iodo; cyano; phenyl; substitutedphenyl; and the like. For substitutions on a phenyl ring, thesubstituents may be in any orientation (i.e., ortho, meta, or para).Preferred non-interfering substituents include lower alkyl, loweralkoxy, cyclopropyl, fluoro, chloro, and cyano.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl (e.g., 0-2substituted phenyl); substituted phenyl; and the like. “Substitutedaryl” is aryl having one or more non-interfering groups as asubstituent. For substitutions on a phenyl ring, the substituents may bein any orientation (i.e., ortho, meta, or para).

As used herein, the “halo” designator (e.g., fluoro, chloro, iodo,bromo, and so forth) is generally used when the halogen is attached to amolecule, while the suffix “ide” (e.g., fluoride, chloride, iodide,bromide, and so forth) is used when the halogen exists in itsindependent ionic form (e.g., such as when a leaving group leaves amolecule).

“Electrophile” refers to an ion or atom or collection of atoms, that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic having a nucleophilic center, i.e., a center that is seeking anelectrophilic center or with an electrophile.

Abasic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

As used herein, the term “ionizable hydrogen atom” (“Ha”) means ahydrogen atom that can be removed in the presence of a base, often ahydroxide or amine base. Typically, the “ionizable hydrogen atom” (“Ha”)will be a hydrogen atom attached to a carbon atom that, in turn, isattached to one or more aromatic moieties or another group or groupsthat in some way stabilize the carbanion that would form from loss ofthe ionizable hydrogen atom as a proton (or the transition state leadingto said carbanion).

As used herein, the term “carboxylic acid” is a moiety having a —C(O)OHfunctional group, as well as moieties that are derivatives of acarboxylic acid, such derivatives including, for example, protectedcarboxylic acids. Thus, unless the context clearly dictates otherwise,the term carboxylic acid includes not only the acid form, butcorresponding esters and protected forms as well. With regard toprotecting groups suited for a carboxylic acid and any other functionalgroup described herein, reference is made to Greene et al., “PROTECTIVEGROUPS IN ORGANIC SYNTHESIS”, 3^(rd) Edition, John Wiley and Sons, Inc.,New York, 1999.

The term “reactive” or “activated” when used in conjunction with aparticular functional group, refers to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

The terms “protected” or “protecting group” or “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive functionalgroup being protected as well as the reaction conditions to be employedand the presence of additional reactive or protecting groups in themolecule, if any. Protecting groups known in the art can be found inGreene et al., supra.

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof. In particular, recitation ofspecific functional groups such as carboxylic acids, aldehydes, orhydroxyl groups encompasses protected forms thereof.

“Multifunctional”, in the context of a polymer of the invention, means apolymer having 3 or more functional groups contained therein, where thefunctional groups may be the same or different. Multifunctional polymersof the invention will typically contain from about 3-100 functionalgroups, or from 3-50 functional groups, or from 3-25 functional groups,or from 3-15 functional groups, or from 3 to 10 functional groups, orwill contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within thepolymer. A “difunctional” polymer means a polymer having two functionalgroups contained therein, either the same (i.e., homodifunctional) ordifferent (i.e., heterodifunctional).

“Branched,” in reference to the geometry or overall structure of apolymer, refers to polymer having 2 or more polymer “arms.” A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, which, for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

A “dendrimer” or dendritic polymer is a globular, size monodispersepolymer in which all bonds emerge radially from a central focal point orcore with a regular branching pattern and with repeat units that eachcontribute a branch point. Dendrimers exhibit certain dendritic stateproperties such as core encapsulation, making them unique from othertypes of polymers.

In the context of the present description, the definition of a variableprovided with respect to one structure or formula is applicable to thesame variable repeated in a different structure, unless the contextdictates otherwise. Thus, for example, the definition of “POLY,” “aspacer moiety,” “R^(e1)” and so forth with respect to a polymer can beequally applicable to a water-soluble polymer conjugate provided herein.

The terms “spacer” or “spacer moiety” (which may also be referred to asa linker or linker moiety) are used herein to refer to an atom or acollection of atoms optionally used to link one moiety to another, suchas a water-soluble polymer segment to a functional moiety in a polymericreagent. The spacer moieties of the invention are preferablyhydrolytically stable but may include one or more physiologicallyhydrolyzable or enzymatically degradable linkages. Exemplary spacermoieties are described further below.

A “physiologically cleavable” or “hydrolyzable” bond is a relativelyweak bond that reacts with water (i.e., is hydrolyzed) underphysiological conditions. The tendency of a bond to hydrolyze in waterwill depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include, butare not limited to, carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, ortho esters, peptides andoligonucleotides.

A “degradable linkage” includes, but is not limited to, aphysiologically cleavable bond, a hydrolyzable bond, and anenzymatically degradable linkage. Thus, a “degradable linkage” is alinkage that may undergo either hydrolysis or cleavage by some othermechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, andso forth) under physiological conditions. For example, a “degradablelinkage” can involve an elimination reaction that has a base abstractionof a proton, (e.g., an ionizable hydrogen atom, Ha), as the drivingforce.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes (carbamates), and the like. Generally, ahydrolytically stable linkage is one that exhibits a rate of hydrolysisof less than about 1-2% per day under physiological conditions.Hydrolysis rates of representative chemical bonds can be found in moststandard chemistry textbooks. It must be pointed out that some linkagescan be hydrolytically stable or hydrolyzable, depending upon (forexample) adjacent and neighboring atoms and ambient conditions. One ofordinary skill in the art can determine whether a given linkage or bondis hydrolytically stable or hydrolyzable in a given context by, forexample, placing a linkage-containing molecule of interest underconditions of interest and testing for evidence of hydrolysis (e.g., thepresence and amount of two molecules resulting from the cleavage of asingle molecule). Other approaches known to those of ordinary skill inthe art for determining whether a given linkage or bond ishydrolytically stable or hydrolyzable can also be used.

As used herein, “drug release rate” means a rate (stated as a half-life)in which half of the total amount of polymer-active agent conjugates ina system will cleave into the active agent and a polymeric residue.

The terms “active agent,” “biologically active agent” and“pharmacologically active agent” are used interchangeably herein and aredefined to include any agent, drug, compound, composition of matter ormixture that provides some pharmacologic, often beneficial, effect thatcan be demonstrated in-vivo or in vitro. This includes foods, foodsupplements, nutrients, nutriceuticals, drugs, proteins, vaccines,antibodies, vitamins, and other beneficial agents. As used herein, theseterms further include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a polymer-active agent conjugate, typicallypresent in a pharmaceutical preparation, that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or ina target tissue. The exact amount will depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the pharmaceutical preparation, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one of ordinary skill in the art, based upon theinformation provided herein and available in the relevant literature.

The term “patient” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as provided herein, and includes both humans and animals.

II. Polymeric Reagents

A. Reactive Structural Features

Polymeric reagents of the invention include, in one aspect, α-hydroxyaldehydes or ketones having the structure I.

where

R¹ is selected from H, lower alkyl, and alkoxyalkyl, and is preferably Hor methyl;

R⁴ is a two- or three-carbon chain which may be substituted with one ormore groups selected from alkyl, alkenyl, aryl, alkoxy, halo, cyano, anda water soluble polymer, wherein the carbon adjacent to Cα is notsubstituted with hydroxy, and wherein two substituents on R⁴ maytogether form an aliphatic or aromatic ring; and

NR⁵ is a secondary or tertiary amino group which is linked to a watersoluble polymer POLY, preferably a polyethylene glycol, via an optionalspacer group Z, where R⁵ is hydrogen or an alkyl group, which may form aring with spacer group Z.

In one embodiment, R¹ is H, such that the reagent contains an α-hydroxyaldehyde. In further embodiments, R⁴ is unsubstituted or is substitutedwith lower alkyl. R⁴ in structure I is preferably a three-carbon chain,and may be saturated or unsaturated. More particularly, reagents inwhich R⁴ is a saturated three-carbon chain may have the structure Ia:

where R¹, NR⁵, Z and POLY are as defined above; each of the substituentsR^(c), R^(c′), R^(d), and R^(d′) is independently selected fromhydrogen, alkyl, alkenyl, aryl, alkoxy, halo, cyano, hydroxy, and awater soluble polymer, and each of the substituents R^(b) and R^(b′) isindependently selected from hydrogen, alkyl, alkenyl, aryl, alkoxy,halo, cyano, and a water soluble polymer; wherein at most one of thesesubstituents is a water soluble polymer, and wherein any two of thesesubstituents can together form an aliphatic ring.

In selected embodiments of this structure, the substituents R^(b),R^(b′), R^(c), R^(c′), R^(d), and R^(d′) are independently selected fromhydrogen and alkyl, wherein any two such alkyl substituents, preferablyon adjacent carbon atoms, can together form a 5- to 7-membered aliphaticring.

In one embodiment, the group R⁵ is methyl. Reagents of this classinclude those designated (5) and (10) herein(5-(mPEG-methyl-amino)-2-hydroxypentanal) (depicted below). In theseexemplary reagents, each of R^(b), R^(b′), R^(c), R^(c′), R^(d), andR^(d′) in structure Ia is hydrogen, and Z is absent.

The spacer group Z, when present, preferably consists of bonds selectedfrom alkylene, ether, thioether, amide, and amine. The spacer group,when present, is typically up to about 15 atoms in length, preferably upto 8 atoms in length. In one embodiment having a spacer group, NR⁵together with Z forms a ring to which POLY is linked, e.g. a piperazinering to which POLY is linked via a ring nitrogen atom. Reagents of thisclass include those designated (11) and (12) herein(5-mPEG-piperazine-2-hydroxy-pentanal) (depicted below). In theseexemplary reagents, each of R^(b), R^(b′), R^(c), R^(c′), R^(d), andR^(d′) in structure Ia is hydrogen.

Analogous reagents include those in which the ring formed by NR⁵together with Z is of a different size (e.g. 5- or 7-membered rings) orcomposition (e.g. comprising only carbon and hydrogen in addition tonitrogen, or including other atoms such as oxygen in the ring).

In another aspect, the invention provides a polymeric α-hydroxy aldehydeor ketone reagent having the structure II:

where

POLY is a water soluble polymer, preferably a poly(ethylene glycol)(PEG), which may include a linker moiety, as described herein, linkingit to (CR²R³)_(m);

R¹ is selected from H, alkyl, hydroxyalkyl, and alkoxyalkyl;

m is 0-12, preferably 0-6; and in selected embodiments 3-6;

each R² and R³ is independently selected from H, alkyl, alkylene,hydroxy, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxyalkylene, aminoalkyl,iminoalkyl, carboxylic acid, alkylcarboxylic acid, phosphate,alkylphosphate, and a further water soluble polymer,

wherein at most one R² or R³ groups is a water soluble polymer, andwherein two substituents R² or R³ in (CR²R³)_(m) can together form aring.

In preferred embodiments, at most one group R² or R³ in the polymericreagent is selected from aminoalkyl, iminoalkyl, carboxylic acid,alkylcarboxylic acid, phosphate, and alkylphosphate. “Aminoalkyl” and“iminoalkyl” can include (cyclic amino)alkyl or (cyclic imino)alkyl;i.e. where the amine nitrogen is included in a ring, which may itselfform part of the —(CR²R³)_(m)— chain.

Preferably, R¹ is selected from H and lower alkyl; in selectedembodiments, R¹ is H or methyl.

A polymeric reagent of structure II may be a polymeric derivative of areducing sugar, such as ribose, xylose, arabinose, lyxose, mannose, orfructose, which are α-hydroxy aldehydes or ketones in their open ringform. Reagents based on the 3-deoxy analogs of reducing sugars, such as3-deoxy ribose, are preferred. One such reagent is the compounddesignated herein as 13 (depicted below with the ribose configuration).

In selected embodiments, the reagents II do not include polymericderivatives of reducing sugars which are not 3-deoxy analogs. That is,in preferred embodiments, the substituents R² and R³ on the carbonadjacent to Cα do not include a 1,2,3-trihydroxypropyl group; nor does—(CR²R³)_(m)— include a backbone 1,2,3-trihydroxypropyl residue adjacentto Cα. More particularly, the reagents do not include polymericderivatives of D-glucose or D-galactose, in particular the 3- or6-ethers of D-glucose or D-galactose with PEG. Such reagents tend toproduce conjugates which are less stable than those produced by the3-deoxy analogs, due to the presence of the 3-hydroxyl group adjacent acarbonyl group in the conjugate.

In still further embodiments, —(CR²R³)_(m)— in structure II is definedby —CH₂—CHR^(x)—C(NH₂)R^(y)—, where R^(x) and R^(y) form a 5- to7-membered aliphatic ring to which POLY is linked. Such reagents includethose illustrated as follows, where n is 0 to 2:

Also provided are polymeric reagents having the structure III:

where

POLY is a water soluble polymer, such as a polyethylene glycol, whichmay include a linker moiety, as described herein, linking it to thearomatic ring; and

(i) R⁷ is —CH(OH)—C(═O)—R¹, and R⁸ is selected from —C(═O)OH, —CH₂OH,—C(═O)N(R¹)₂, —CH₂N(R¹)₂, —OH, and —N(R¹)₂, and is preferably selectedfrom —C(═O)OH, —CH₂OH, —C(═O)N(R¹)₂, and —CH₂N(R¹)₂; or

(ii) R⁷ is —CHR¹CH(OH)—C(═O)—R¹, and R⁸ is —OH or —N(R¹)₂;

where each R¹ is independently selected from H, lower alkyl, andalkoxyalkyl, and is preferably independently selected from H and methyl.In selected embodiments, R¹ attached to C(═O) is H, such that thereagent contains an α-hydroxy aldehyde. In other embodiments, each R¹ isH.

Exemplary reagents of this class include the compounds designated hereinas 6, where R⁷ is —CH(OH)—C(═O)—H and R⁸ is —C(═O)OH; 7, where R⁷ is—CH(OH)—C(═O)—H and R⁸ is —OH; and 8, where R⁷ is —CH₂CH(OH)—C(═O)—H andR⁸ is —OH (shown below in protected form, with attached mPEG polymers).Analogous reagents include those in which the phenolic hydroxy group(OH) is replaced by NH₂ or N(CH₃)₂ (that is, R⁸ is —NH₂ or —N(CH₃)₂).

As noted above, the POLY component of all the disclosed reagents is awater soluble polymer, preferably a poly(ethylene glycol). The POLYcomponent of the reagent, and of the resulting conjugates, is discussedin further detail below. The reagents typically have from one to threeattached polymers per molecule, and more typically one or two. In oneembodiment, a reagent has one attached polymer per molecule.

It should be noted that the invention encompasses protected (typicallyacetal and ketal) forms of the aldehyde and ketone in the structuresrepresented herein; for example, C═O can be replaced by C(OR)₂, where Ris alkyl, and the two R groups constitute two monovalent alkyl groups orone bivalent alkyl group (i.e. a cyclic protecting group).

B. Reactivity and Self-Catalysis

The polymeric α-hydroxy aldehyde or α-hydroxy ketone reagents describedherein can be conjugated to compounds containing a reactive amine groupto form a stable conjugate. The reaction takes place in a single step;that is, no reductive step is needed to convert the initial product to astable derivative, as in the widely employed reductive amination ofaldehydes and ketones.

In reactions of the current reagents with amine-containing compounds,the initial conjugation product undergoes an acid- or base-catalyzedrearrangement to an aminoketone, as shown in the second step below. Aversion of this rearrangement was first reported by Amadori, in theconversion of N-glycosides of aldoses to N-glycosides of thecorresponding ketoses.

The rearrangement step which immediately follows conjugation using thecurrent reagents is acid- or base-catalyzed, as noted above.Accordingly, the reaction is preferably carried out in the presence ofanionic buffer salts, such as carbonate and phosphate.

In one embodiment, the polymeric reagent itself incorporates an internalproton-abstracting (basic) functional group, to promote more efficientrearrangement of the imine intermediate. Substituents that provide thisfunctional group may include carboxylates (e.g. aspartic acidderivatives), phosphates, hydroxyl groups, and amines or imines, cyclicas well as acyclic. The substituent is appropriately situated, via alinker if necessary, to position the group for proton abstraction,preferably providing a 4- or 5-bond spacing between the abstracting atomand the hydrogen atom on the α-carbon (that is, a 3- or 4-bond spacingbetween the abstracting atom and the α-carbon itself).

The self-catalysis mechanism is illustrated below, showing the preferred5-bond spacing, such that the transition state represents a 6-memberedring. The proton-abstracting atom is represented by X in this structure.

Preferred self-catalyzing reagents include reagents of structures I/Iaand III above, as well as reagents of structure II above in which onegroup R² or R³ is selected from hydroxy, alkoxy, hydroxyalkyl,alkoxyalkyl, alkoxyalkylene, amino, aminoalkyl, iminoalkyl, carboxylicacid, alkylcarboxylic acid, phosphate, and alkylphosphate; and the groupR² or R³ and the value of m are selected such that an oxygen or nitrogenatom on R² or R³ is separated from Cα by a four-bond path (or, lesspreferably, a three-bond path). Preferably, in such reagents, one groupR² or R³ is selected from amino, aminoalkyl, iminoalkyl, carboxylicacid, and alkylcarboxylic acid.

Self-catalyzing reagents can also be represented by structure VII below:

where:

R¹ is selected from H, lower alkyl, and alkoxyalkyl;

R⁴ and R⁵ are carbon atoms, each of which is substituted with two groupsselected from hydrogen, alkyl, alkenyl, alkoxy, a non-interferingsubstituent as defined herein (e.g. halo, cyano, aryl or substitutedaryl), and a water soluble polymer; Y is carbon, X is nitrogen oroxygen, and R⁶ is selected from an electron pair, hydrogen, alkyl,alkenyl, alkoxy, and a water soluble polymer; such that (a) Y—X—R⁶represents a carboxyl, alkoxylalkyl, aminoalkyl, or iminoalkyl(ene)moiety, where Y—X may be part of a cyclic structure in the case ofaminoalkyl or iminoalkyl(ene), or (b) X—R⁶ taken alone represents aminoor hydroxyl;

wherein R⁴ and R⁵ taken together, or R⁵ and Y taken together, or R⁴ andY taken together when R⁵ is absent, may form one side of a five- orsix-membered ring which may be substituted with a water soluble polymer;

and wherein the reagent comprises at least one water soluble polymer,and typically comprises at most one or two water soluble polymers.

Preferred structures where R⁵ is present (subscript=1) provide asix-membered transition state for the rearrangement, as illustratedabove. Structures in which R⁵ is absent (subscript=0) provide afive-membered transition state for the rearrangement and can also beuseful.

Preferably, R¹ is selected from H and lower alkyl; in selectedembodiments, R¹ is H or methyl. When R⁴ and R⁵, or R⁵ and Y, form oneside of a five- or six-membered ring, such a ring may be furthersubstituted with one or more non-interfering substituents as definedabove.

In one embodiment, Y—X in structure VII represents aminoalkyl, and hasthe structure —CH₂—NR—, where R is lower alkyl. A reagent of this typein which R⁶ is PEG, R is methyl, and each of R⁴ and R⁵ is —CH₂— isdesignated herein as PEG-MAHP-ALD (5 or 10) (where MAHP indicatesmethylamino 2-hydroxy pentanal) and is used in the working Examplesbelow. This reagent is also an embodiment of structure I/Ia, as notedabove.

In another embodiment, Y—X in structure VII represents a carboxyl group(—C(═O)—O—) and R⁶ is hydrogen, giving a carboxylic acid(COOH)-substituted reagent. A reagent of this type in which R¹ ishydrogen, and R⁴ and R⁵ form one side of a benzene ring which issubstituted with mPEG is shown (in aldehyde-protected form) below:

This reagent is also an embodiment of structure III, as noted above.

In a further embodiment, in which Y—X—R⁶ represents an iminoalkyl(ene)moiety, Y—X in structure VII represents a 4-imidazoyl group (anembodiment of a cyclic imino group). A reagent of this type in which R⁴is substituted with hydrogen and —Z-PEG, where Z is an amide linker, andR⁵ is —CH₂—, is shown below. (Preparation is described in Example 3.)This reagent is also an embodiment of structure II above, where R² or R³is (cyclic imino)alkyl.

In a further embodiment, X—R⁶ taken alone represents a hydroxyl group,and R⁵ and Y taken together form one side of a six-membered ring, suchas a benzene ring, which is substituted with a water soluble polymer. Areagent of this type, where R⁴ is —CH₂—, is shown (in protected form) onthe right below (structure 8).

As noted above, the reagents of structure VII where R⁵ is present(subscript=1) are designed to provide a favorable six-memberedtransition state for the rearrangement. Also useful are structuresdesigned to provide a five-membered transition state; these wouldessentially have the structure VII with R⁵ omitted (subscript=0), sothat R⁴ is directly connected to Y, as shown below (structure VII′). Inthis case, as noted above, R⁴ and Y taken together may form one side ofa five- or six-membered ring which may be substituted with a watersoluble polymer. An example of this class of reagent is shown (inprotected form) in the structure on the left above (structure 7).

Reagents 7 and 8 are also embodiments of structure III, as noted above.

The polymeric reagents are prepared according to standard syntheticmethods available to those skilled in the art. For example, a precursorcompound having a protected aldehyde or ketone at one terminus and areactive group at another terminus is reacted with a polymer having acomplementary reactive group. Alternatively, the aldehyde or ketone maybe generated following attachment of the polymer to a suitable precursormolecule. See, for example, the schemes illustrated in Examples 1-5,7-10 and 18-19 below, which are not to be regarded as limiting.

C. The Water Soluble Polymer

With respect to a given water-soluble polymer in the presently describedpolymeric reagents, each water-soluble polymer (e.g., POLY¹, POLY² etc.)can comprise any polymer so long as the polymer is water-soluble.Moreover, a water-soluble polymer as used herein is typicallynon-peptidic.

Although preferably a poly(ethylene glycol), a water-soluble polymer foruse herein can be, for example, other water-soluble polymers such asother poly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers of ethylene glycol and propylene glycol and the like,poly(olefinic alcohol), poly(vinylpyrrolidone),poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate),poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol),polyphosphazene, polyoxazoline, poly(N-acryloylmorpholine), such asdescribed in U.S. Pat. No. 5,629,384. The water soluble polymer can be ahomopolymer, copolymer, terpolymer, nonrandom block, and random blockpolymer of any of the foregoing.

Thus, the water soluble polymer preferably comprises up to threedifferent monomers selected from the group consisting of: alkyleneglycol, such as ethylene glycol or propylene glycol; olefinic alcohol,such as vinyl alcohol, 1-propenol or 2-propenol; vinyl pyrrolidone;hydroxyalkyl methacrylamide or hydroxyalkyl methacrylate, where alkyl ispreferably methyl; saccharide; α-hydroxy acid, such as lactic acid orglycolic acid; phosphazene, oxazoline, and N-acryloylmorpholine.Preferred monomer types include alkylene glycol, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid.

Preferably, the polymer is a copolymer of two monomers selected fromthis group, or, more preferably, a homopolymer of one monomer selectedfrom this group. Thus, representative POLYs include poly(alkyleneglycols) such as poly(ethylene glycol), poly(propylene glycol) (“PPG”),copolymers of ethylene glycol and propylene glycol, poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline, andpoly(N-acryloylmorpholine). POLY can be a homopolymer, an alternatingcopolymer, a random copolymer, a block copolymer, an alternatingtripolymer, a random tripolymer, or a block tripolymer of any of theabove. The two monomers in a copolymer may be of the same monomer type,for example, two alkylene glycols, such as ethylene glycol and propyleneglycol.

In instances where the polymeric reagent comprises two or morewater-soluble polymers, each water-soluble polymer in the overallstructure can be the same or different. It is preferred, however, thatall water-soluble polymers in the overall structure are of the sametype. For example, it is preferred that all water-soluble polymerswithin a given structure are each a poly(ethylene glycol).

Any water-soluble polymer having at least one reactive terminus can beused to prepare a polymeric reagent in accordance with the invention.Although water-soluble polymers bearing only a single reactive terminuscan be used, polymers bearing two, three, four, five, six, seven, eight,nine, ten, eleven, twelve or more reactive termini suitable forconversion as set forth herein can be used. In the context of beingpresent within an overall structure, a water-soluble polymer has from 1to about 300 termini.

The polymer component can have any of a number of different geometries;for example, POLY can be linear, branched, or forked, as will bedescribed in further detail below. Most typically, POLY is linear or isbranched, for example, having 2 polymer arms.

Although the weight average molecular weight of any individualwater-soluble polymer can vary, the weight average molecular weight ofany given water-soluble polymer will typically be in the followingrange: 100 Daltons to about 150,000 Daltons. Exemplary ranges, however,include weight-average molecular weights in the range of about 880Daltons to about 5,000 Daltons, in the range of greater than 5,000Daltons to about 100,000 Daltons, in the range of from about 6,000Daltons to about 90,000 Daltons, in the range of from about 10,000Daltons to about 85,000 Daltons, in the range of greater than 10,000Daltons to about 85,000 Daltons, in the range of from about 20,000Daltons to about 85,000 Daltons, in the range of from about 53,000Daltons to about 85,000 Daltons, in the range of from about 25,000Daltons to about 120,000 Daltons, in the range of from about 29,000Daltons to about 120,000 Daltons, in the range of from about 35,000Daltons to about 120,000 Daltons, in the range of about 880 Daltons toabout 60,000 Daltons, in the range of about 440 Daltons to about 40,000Daltons, in the range of about 440 Daltons to about 30,000 Daltons, andin the range of from about 40,000 Daltons to about 120,000 Daltons. Forany given water-soluble polymer, PEGs having a molecular weight in oneor more of these ranges are preferred.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 440 Daltons, about 500 Daltons, about 600 Daltons,about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons,about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons,about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000Daltons, about 14,000 Daltons, about 15,000 Daltons, about 16,000Daltons, about 17,000 Daltons, about 18,000 Daltons, about 19,000Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000Daltons, and about 75,000 Daltons. Branched versions of thewater-soluble polymer (e.g., a branched 40,000 Dalton water-solublepolymer comprised of two 20,000 Dalton polymers) having a total weightaverage molecular weight of any of the foregoing can also be used.

When a PEG is used as the water-soluble polymer in the polymericreagent, the PEG typically comprises a number of (OCH₂CH₂) monomers (or(CH₂CH₂O) monomers, depending on how the PEG is defined). As usedthroughout the description, the number of repeating units is identifiedby the subscript “n” in “(OCH₂CH₂)_(n).” Thus, the value of (n)typically falls within one or more of the following ranges: from 2 toabout 3400, from about 100 to about 2300, from about 100 to about 2270,from about 136 to about 2050, from about 225 to about 1930, from about450 to about 1930, from about 1200 to about 1930, from about 568 toabout 2727, from about 660 to about 2730, from about 795 to about 2730,from about 795 to about 2730, from about 909 to about 2730, and fromabout 1,200 to about 1,900. For any given polymer in which the molecularweight is known, it is possible to determine the number of repeatingunits (i.e., “n”) by dividing the total weight-average molecular weightof the polymer by the molecular weight of the repeating monomer.

Each water-soluble polymer is typically biocompatible andnon-immunogenic. With respect to biocompatibility, a substance isconsidered biocompatible if the beneficial effects associated with useof the substance alone or with another substance (e.g., an active agent)in connection with living tissues (e.g., administration to a patient)outweighs any deleterious effects as evaluated by a clinician, e.g., aphysician. With respect to non-immunogenicity, a substance is considerednon-immunogenic if use of the substance alone or with another substancein connection with living tissues does not produce an immune response(e.g., the formation of antibodies) or, if an immune response isproduced, that such a response is not deemed clinically significant orimportant as evaluated by a clinician. It is particularly preferred thatthe polymers and water-soluble polymer segments, described herein aswell as conjugates of active agents and the polymers are biocompatibleand non-immunogenic.

In preferred embodiments, POLY is a poly(ethylene glycol). PEG polymersare typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, and non-toxic, and do not hydrolyzeor deteriorate (unless specifically designed to do so). Poly(ethyleneglycol) is highly biocompatible and substantially non-immunogenic. Whenconjugated to a pharmacologically active agent, the PEG molecule tendsto mask the agent and can reduce or eliminate any immune response to theagent. PEG conjugates tend not to produce a substantial immune responseor cause clotting or other undesirable effects.

The molecular weight of the PEG component may vary, as described above,but preferred polymers include from about 3 to about 4,000, or fromabout 3 to about 3,000, or more preferably from about 20 to about 1,000monomeric units.

In one form, free or nonbound PEG is a linear polymer terminated at eachend with hydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OHwherein (m′) typically ranges from zero to about 4,000, preferably fromabout 20 to about 1,000.

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH, where it is understood that the-PEG- symbol can represent the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—

where (m′) is as defined as above.

An end-capped PEG, where PEG is terminally capped with an inertend-capping group, is preferred for preparing the reagents of theinvention. Preferred end-capping moieties include alkoxy, substitutedalkoxy, alkenyloxy, substituted alkenyloxy, alkynyloxy, substitutedalkynyloxy, aryloxy, and substituted aryloxy. Most preferred end-cappinggroups are methoxy, ethoxy, and benzyloxy. The structure of mPEG(methoxy-PEG-OH) is given below:CH₃₀—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OH

where (m′) is as described above.

The capping group may also be a phospholipid. A preferred phospholipidis a dialkyl phosphatidylethanolamine, such as distearoylphosphatidylethanolamine (DSPE; see structure below). Other suitablephospholipids include, for example, phosphatidyl serines, phosphatidylglycerols, phosphatidyl inositols, and phosphatidyl cholines, all ofwhich are well known in the art and available commercially. A reactivegroup on the phosphate head group can be used to link the lipid to a PEGchain; for example, the terminal amine in DSPE can be linked to PEG viaa carbamate linkage.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages.

In a preferred embodiment, the branched PEG polymer is methoxypoly(ethylene glycol) disubstituted lysine. Invention compound 14 isanother example of a reagent utilizing a branched PEG.

In addition, the PEG can comprise a forked PEG. An example of a free ornonbound forked PEG is represented by the following structure:

where X is a spacer moiety and each Z is an activated terminal grouplinked to CH by a chain of atoms of defined length. InternationalApplication Pubn. No. WO 99/45964 discloses various forked PEGstructures capable of use in the present invention. The chain of atomslinking the Z functional groups to the branching carbon atom serve as atethering group and may comprise, for example, alkyl chains, etherchains, ester chains, amide chains and combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, each water-solublepolymer in the polymeric reagent can also be prepared with one or moreweak or degradable linkages in the polymer, including any of the abovedescribed polymers. For example, PEG can be prepared with ester linkagesin the polymer that are subject to hydrolysis. As shown below, thishydrolysis results in cleavage of the polymer into fragments of lowermolecular weight:-PEG-CO₂-PEG-+H₂O

-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, and a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by, for example,a phosphoramidite group, e.g., at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the above formsof PEG.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning substantially water-soluble polymers is by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the term “water-soluble polymer” refers both to a molecule aswell as the residue of water-soluble polymer that has been attached toanother moiety.

D. Linker Moieties

A linker moiety, or simply “linker”, may be used to connect polymersegments making up the component POLY to each other, or to connect thepolymer component POLY to the functional portion of the reagent. Theterm POLY as used herein is understood to optionally include such alinker. A linker may be a single atom, such as an oxygen or a sulfur,two atoms, or a number of atoms. A linker is typically but is notnecessarily linear in nature. The overall length of the linker willtypically range between 1 to about 40 atoms, where by length is meantthe number of atoms in a single chain, not counting substituents. Forinstance, —CH₂— counts as one atom with respect to overall linkerlength, and —CH₂CH(CH₃)O— counts as 3 atoms in length. Preferably, alinker will have a length of about 1 to about 20 atoms, or, morepreferably, from about 2 to about 15 atoms.

A linker can be a single functional group such as an amide, an ester, aurethane (carbamate), or a urea, or it may contain methylene or otheralkylene groups flanking either side of the single functional group.Alternatively, a linker may contain a combination of functional groups,which can be the same or different. A linker can be an alkylene chain,optionally containing one or more oxygen or sulfur atoms (i.e., an etheror thioether linkage). Also included are alkylene chains containing anitrogen atom (i.e. an amine linkage.) Preferred linkers are those thatare hydrolytically stable.

Illustrative linkers are those corresponding to either of the followingstructures:

—(CH₂)_(c)-D_(c)-(CH₂)_(f)— or —(CH₂)_(p)-M_(r)-C(O)—K_(s)—(CH₂)_(q)—.In referring to these structures, the variable “c” ranges from zero to8; “D” is O, NH, or S; the variable “e” is 0 or 1; the variable “f”ranges from zero to 8; the variable “p” ranges from zero to 8; “M” is—NH or O; “K” is NH or O; the variable “q” ranges from zero to 8, andthe variables “r” and “s” are each independently 0 or 1.

Exemplary linker moieties include, but are not limited to, thefollowing: —O—, —S—, —C(O)—, —S(O₂)—, —S(O)—, —NH—S(O₂)—, —S(O₂)—NH—,—CH═CH—, —O—CH═CH—, —C(O)—NH—, —NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—,—CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂, —O—CH₂—, —CH₂—O—,—O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —S—CH₂—, —CH₂—S—, —S—CH₂—CH₂—, —CH₂—S—CH₂—,—CH₂—CH₂—S—, —S—CH₂—CH₂—CH₂—, —CH₂—S—CH₂—CH₂—, —CH₂—CH₂—S—CH₂—,—CH₂—CH₂—CH₂—S—, —S—CH₂—CH₂—CH₂—CH₂—, —CH₂—S—CH₂—CH₂—CH₂—,—CH₂—CH₂—S—CH₂—CH₂—, —CH₂—CH₂—CH₂—S—CH₂—, —CH₂—CH₂—CH₂—CH₂—S—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—C(O)—NH—,—NH—C(O)—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH₂—CH₂—CH₂—C(O)—NH—,—NH—C(O)—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —NH—C(O)—CH═CH—C(O)—NH—,—C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—, —CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—,—NH—C(O)—CH₂—, —CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—,—NH—C(O)—CH₂—CH₂—, —CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂,—NH—CH₂—, —NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—, bivalent cycloalkyl, andamino acids.

Also included are —N(R⁶)—, where R⁶ is H or an organic radical selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl and substitutedaryl; and —NH—C(O)—O—(CH₂)_(h)—(OCH₂CH₂)_(j)— or—O—C(O)—NH—(CH₂)_(h)—(OCH₂CH₂)_(j)—, where (h) is zero to six, and (j)is zero to 20. Other specific spacer moieties have the followingstructures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, —NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—,and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)—, wherein the subscript values followingeach methylene indicate the number of methylenes contained in thestructure, e.g., (CH₂)₁₋₆ means that the structure can contain 1, 2, 3,4, 5 or 6 methylenes.

A linker may include combinations of two or more of any of theforegoing.

Additionally, any of the above spacer moieties may further include anethylene oxide oligomer chain comprising 1 to 20 ethylene oxide monomerunits (i.e., —(CH₂CH₂O)₁₋₂₀). That is, the ethylene oxide oligomer chaincan occur before or after the spacer moiety, and optionally in betweenany two atoms of a spacer moiety comprised of two or more atoms.However, the oligomer chain would not be considered part of the spacermoiety if the oligomer is adjacent to a polymer segment and merelyrepresents an extension of the polymer segment. For example, where POLYis defined as CH₃O(CH₂CH₂O)_(n)—, —CH₂CH₂O— would not be a linkermoiety, since such a definition would merely represent an extension ofthe polymer. However, a linker could contain one or more —CH₂CH₂O—subunits flanked by one or a combination of illustrative linkers asprovided above.

Preferably, the linker is hydrolytically stable, and may contain one ormore of the following functional groups: amide, urethane, ether,thioether, or urea. However, hydrolytically degradable linkages, such ascarboxylate ester, phosphate ester, orthoester, anhydride, imine,acetal, ketal, oligonucleotide, or peptide, may also be present.

III. Continuation Method

In accordance with the invention, methods of forming stable conjugatesof water-soluble polymers with amine-containing biologically activemolecules are provided. As noted above, the reaction takes place in asingle step; that is, no reductive step is needed to convert the initialproduct to a stable derivative, as in the widely employed reductiveamination of aldehydes and ketones.

Accordingly, the invention provides a method of conjugating a watersoluble polymer with a compound having a reactive amine group, themethod comprising: reacting the compound with a polymeric reagentcomprising an α-hydroxy aldehyde or a terminal α-hydroxy ketone,respectively, linked to a water soluble polymer, thereby forming aconjugate in which the water soluble polymer is linked to a moietyhaving the structure —C(═O)—CHR¹—NH—B, where R¹ is hydrogen or methyl,respectively, and NH—B represents the residue of the amine-containingcompound.

In preferred embodiments, the polymeric reagent further comprises aproton-abstracting functional group containing a proton-abstracting atomselected from oxygen and nitrogen, situated such there is a 3- or 4-bondspacing, preferably a 4-bond spacing, between the proton-abstractingatom and the α-carbon of the α-hydroxy aldehyde or α-hydroxy ketone.Such reagents are able to undergo a “self-catalyzed” conjugationreaction, as described above.

More particularly, the invention provides a method of conjugating awater soluble polymer with a compound having a reactive amine group, byreacting the amine-containing compound with a polymeric reagent havingthe structure I/Ia, II or III as shown herein, where the reagentcomprises at least one water soluble polymer; to form a conjugate havingstructure IV, V or VI as described herein, respectively, wherein —NH—Bin structure IV, V or VI represents the residue of the amine-containingcompound.

The conjugation method may employ a polymeric derivative of a reducingsugar, such as ribose, xylose, arabinose, lyxose, galactose, glucose,mannose, and fructose, which are α-hydroxy aldehydes or ketones in theiropen ring form. Reagents based on the 3-deoxy analogs of reducingsugars, such as 3-deoxy ribose, are preferred. Such reagents areadvantageous over the corresponding derivatives of the native sugars,because the absence of the 3-hydroxyl group in the eventual conjugatedproduct reduces opportunities for degradation of the conjugated product.

In general, preferred embodiments of the method employ preferredembodiments of the reagents as described herein. The amine-containingcompound is typically a biologically active or biologically relevantcompound, as described further below.

A. Reaction Conditions

Suitable solvents for carrying out the conjugation reaction includebuffers such as aqueous sodium phosphate, sodium acetate, sodiumcarbonate, phosphate buffered saline (PBS), sodium borate, andN-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES). Forconjugation to a protein, the polymeric reagent is typically added tothe protein-containing solution at an equimolar amount or at a molarexcess relative to target protein. Molar excesses of PEG-reagentrelative to target protein are typically in the range of about 2 to 5.The conjugation reaction is typically carried out at temperatures at orbelow about room temperature (25° C.), although temperatures may rangefrom about −15° C. to about 100° C., more preferably from about 4° C. to37° C., for approximately one to 24 hours. Exemplary conjugationreaction conditions are described in Examples 6 and 11-13 below.

Conditions for conjugation to a small molecule, e.g. amphotericin B orother amine-containing molecules as discussed below, will vary accordingto the small molecule being modified. Typically, however, theconjugation is conducted using a slight molar excess of polymericreagent relative to small molecule, e.g., about 1.2-1.5, to about a 5 to10-fold molar excess. In some instances, depending upon the molecule,the small molecule drug may actually be used in excess, such as when thePEG-small molecule conjugate precipitates in the reaction solvent, e.g.,ether, while the unreacted drug remains in solution.

The exact reaction time is determined by monitoring the progress of thereaction over time. Progress of the reaction is typically monitored bywithdrawing aliquots from the reaction mixture at various time pointsand analyzing the reaction mixture by SDS-PAGE or MALDI-TOF massspectrometry or any other suitable analytical method.

B. Characterization and Optional Separation of PEG-Mers

Optionally, conjugates produced by reacting a PEGylated reagent of theinvention with a biologically active agent are purified toobtain/isolate different PEGylated species, when the conjugated molecule(e.g., a protein) and reaction conditions are such that multiplePEGylated species are to be expected. For reaction of proteins withlower molecular weight PEGs, e.g., having molecular weights less thanabout 20 kilodaltons, the reaction product can be purified to obtain adistribution around a certain number of PEGs per protein molecule. Forexample, the product mixture can be purified to obtain an average ofanywhere from one to five PEGs per protein, typically an average ofabout 3 PEGs per protein. The strategy for purification of the finalconjugate reaction mixture will depend upon a number of factors, such asthe molecular weight of the polymer employed, the particular protein,and the residual activity and in vivo properties of the individualconjugate species.

PEG conjugates having different molecular weights can be isolated usinggel filtration chromatography. For example, in an exemplary reactionwhere a PEG reagent having a molecular weight of about 20 kDa israndomly conjugated to a 100 kDa protein, the resulting reaction mixturewill likely contain unmodified protein (MW 100 kDa), monopegylatedprotein (MW 120 kDa), di-pegylated protein (MW 140 kDa), etc. Gelfiltration columns suitable for carrying out this type of separationinclude Superdex© and Sephadex© columns available from AmershamBiosciences. Selection of a particular column will depend upon thedesired fractionation range desired. Elution is generally carried outusing a non-amine based buffer, such as phosphate, acetate, or the like.The collected fractions may be analyzed by a number of differentmethods, for example, (i) OD at 280 nm for protein content, (ii) BSAprotein analysis, (iii) iodine testing for PEG content, or (iv) byrunning an SDS PAGE gel, followed by staining with barium iodide.

Separation of positional isomers (i.e. conjugates having PEG attached todifferent locations on a protein), generally not achievable by molecularweight-based methods, can be carried out by reverse phase chromatographyusing e.g. an RP-HPLC C18 column (Amersham Biosciences or Vydac).

C. Conjugation to Proteins: Random and N-Terminal Selective

Generally, the polymeric reagents of the invention can be used toselectively target the modification of the N-terminus of a protein,under conditions that differentiate the reactivity of the α-amine at theN-terminal amino acid. Reaction conditions for preparing an N-terminallymodified protein or peptide include (i) dissolving the protein orpeptide to be modified in a non-amine-containing buffer (e.g., at a pHrange from about 4 to about 6.5, preferably from about 5 to 6.5, mostpreferably at a pH of about 5 to 5.5), (ii) adding to the protein orpeptide solution a polymeric reagent (α-hydroxy aldehyde or ketone) ofthe invention, and (iii) allowing the protein or peptide and polymericreagent to react to form the conjugate.

Reaction under conditions of higher pH can be used for random attachmentof a polymeric reagent (α-hydroxy aldehyde or ketone). Morespecifically, to covalently attach a polymeric reagent to any number oflysine residues that are surface accessible, a protein or peptide (suchas those exemplary biomolecules provided below) is typically reactedwith a polymeric reagent of the invention in a non amine-containingbuffer at mild pH, generally ranging from about 5 to 8, more preferablyfrom about 6.5 to 8. Non-amine containing buffers are preferred, sincethe amino-groups in the buffer can compete with protein amino groups forcoupling to the polymeric reagent. A suitable non-amine containingbuffer is selected having an appropriate pK for the desired pH range forconducting the conjugation chemistry. The coupling reaction generallytakes anywhere from minutes to several hours (e.g., from 5 minutes to 24hours or more), and on average, coupling is achieved between about 0.2and 4 hours.

The degree of modification, that is, the number of PEGs that arecovalently attached at available sites on the target molecule, can beincreased by increasing, either independently or simultaneously, any oneor more of: molar ratio of polymeric reagent to protein or peptide,temperature, reaction time, and pH.

IV. Polymeric Conjugates

As described above, the present invention provides polymeric reagentsuseful in providing conjugates having a degradable linkage between apolymer and another moiety. Without wishing to be bound by theory, it isbelieved that the conjugates degrade in such as way as to minimize oreliminate entirely any residue or “tag” of the polymeric reagent used toform the conjugate. As a consequence, it is possible, upon hydrolysis ofa conjugate formed from the reaction of a polymeric reagent describedherein with an amine-containing active agent, to regenerate or recoverthe original unconjugated and unmodified form of the active agent.

In accordance with the invention, polymeric conjugates, comprising awater soluble polymer covalently linked to a biologically activemolecule via an α-amino ketone linkage, are provided. The conjugates areprepared by reaction of a reagent of structure I/Ia, II or III hereinwith an amine-containing compound.

In accordance with reagents having the structure I/Ia above, suchconjugates may have the structure IV:

where

R¹ is selected from H, lower alkyl, and alkoxyalkyl, and is preferably Hor methyl;

R⁴ is a two- or three-carbon chain which may be substituted with one ormore groups selected from alkyl, alkenyl, aryl, alkoxy, halo, cyano, anda water soluble polymer; wherein the carbon adjacent to the carbonylcarbon is not substituted with hydroxyl, and wherein two substituents onR⁴ may form an aliphatic or aromatic ring;

NR⁵ is a secondary or tertiary amino group which is linked to a watersoluble polymer POLY, such as a polyethylene glycol, via an optionalspacer group Z, where R⁵ is hydrogen or an alkyl group, which may form aring with spacer group Z; and

—NH—B represents the residue of an amine-containing biologically activecompound.

In accordance with reagents having the structure II above, suchconjugates may also have the structure V:

wherein:

POLY is a water soluble polymer, preferably a PEG, which may include alinker moiety, as described herein, linking it to (CR²R³)_(m);

R¹ is selected from H, alkyl, hydroxyalkyl, and alkoxyalkyl, andpreferably from H and methyl;

m is 0-12, preferably 0-6, and in selected embodiments 3-6; and

each R² and R³ is independently selected from H, alkyl, alkylene,hydroxy, amino, alkoxy, hydroxyalkyl, alkoxyalkyl, alkoxyalkylene,aminoalkyl, iminoalkyl, carboxylic acid, alkylcarboxylic acid,phosphate, alkylphosphate, and a further water soluble polymer, whereinat most one R² or R³ group is a water soluble polymer, and wherein twosubstituents R² or R³ on (CR²R³)_(m) may form a ring, preferably analiphatic ring; and

—NH—B represents the residue of an amine-containing biologically activecompound.

In preferred embodiments, at most one group R² or R³ is selected fromaminoalkyl, iminoalkyl, carboxylic acid, alkylcarboxylic acid,phosphate, and alkylphosphate. As noted above, aminoalkyl and iminoalkylcan include (cyclic amino)alkyl or (cyclic imino)alkyl; i.e. where theamine nitrogen is included in a ring, which may itself form part of the—(CR²R³)_(m)— chain.

Further conjugates of the invention include those of structure VI:

where

(i) R⁷ is C(═O)—CHR¹—NH—B, and R⁸ is selected from —C(═O)OH, —CH₂OH,—C(═O)N(R¹)₂, —CH₂N(R¹)₂, —OH, and —N(R¹)₂, and is preferably selectedfrom —C(═O)OH, —CH₂OH, —C(═O)N(R¹)₂, and —CH₂N(R¹)₂; or

(ii) R⁷ is —CHR¹—C(═O)—CHR¹—NH—B, and R¹ is —OH or —N(R¹)₂; POLY is awater soluble polymer, such as a PEG, which may include a linker moiety,as described herein, linking it to the aromatic ring; and —NH—Brepresents the residue of an amine-containing biologically activecompound; where each R¹ is independently selected from H, lower alkyl,and alkoxyalkyl, and preferably from H and methyl.

In general, preferred embodiments of the conjugates IV, V and VI arederived from preferred embodiments of the corresponding reagents I/Ia,II and III as described herein.

In particularly preferred embodiments, the conjugates are derived fromself-catalyzing reagents. These include conjugates of structures IV andVI above, as well as conjugates of structure V above in which one groupR² or R³ is selected from hydroxy, alkoxy, amino, hydroxyalkyl,alkoxyalkyl, alkoxyalkylene, aminoalkyl, iminoalkyl, carboxylic acid,alkylcarboxylic acid, phosphate, and alkylphosphate; and this group R²or R³ and the value of m are selected such that an oxygen or nitrogenatom on R² or R³ is separated from Cα by a four-bond path. Preferably,R² or R³ is selected from aminoalkyl, iminoalkyl, carboxylic acid, andalkylcarboxylic acid.

Certain conjugates derived from self-catalyzing reagents can also berepresented by the structure VIII (derived from reagent structure VIIabove):

where

R¹ is selected from H, lower alkyl, and alkoxyalkyl;

R⁴ and R⁵ are carbon atoms, each of which is substituted with two groupsselected from hydrogen, alkyl, alkenyl, alkoxy, a non-interferingsubstituent (e.g. halo, cyano, aryl or substituted aryl) and a watersoluble polymer; Y is carbon, X is nitrogen or oxygen, and R⁶ isselected from an electron pair, hydrogen, alkyl, alkenyl, alkoxy, and awater soluble polymer; such that (a) Y—X—R⁶ represents a carboxyl,alkoxylalkyl, aminoalkyl, or iminoalkyl(ene) moiety, where Y—X may bepart of a cyclic structure in the case of aminoalkyl or iminoalkyl(ene),or (b) X—R⁶ taken alone represents amino or hydroxyl;

wherein R⁴ and R⁵ taken together, or R⁵ and Y taken together, or R⁴ andY taken together when R⁵ is absent, may form one side of a five- orsix-membered ring which may be substituted with a water soluble polymer;

and wherein the reagent comprises at least one water soluble polymer;

and —NH—B represents the residue of an amine-containing biologicallyactive compound.

Preferred embodiments of such conjugates include those corresponding toembodiments described for reagent structure VII, above.

V. The Conjugated Amine-Containing Agent

The biologically active agent conjugated to a polymeric reagent of theinvention may fall into one of a number of structural classes, includingbut not limited to small molecules (including difficultly soluble smallmolecules), peptides, polypeptides, proteins, polysaccharides, steroids,nucleotides, oligonucleotides, polynucleotides, fats, electrolytes, andthe like. The molecule either possesses a native amino group or ismodified to contain at least one reactive amino group suitable forcoupling to a polymeric reagent of the invention.

The agent may be a therapeutic substance selected from, for example,hypnotics and sedatives, psychic energizers, tranquilizers, respiratorydrugs, anticonvulsants, muscle relaxants, antiparkinson agents (dopamineantagonists), analgesics, anti-inflammatories, antianxiety drugs(anxiolytics), appetite suppressants, antimigraine agents, musclecontractants, anti-infectives (antibiotics, antivirals, antifungals,vaccines) antiarthritics, antimalarials, antiemetics, anepileptics,bronchodilators, cytokines, growth factors, anti-cancer agents,antithrombotic agents, antihypertensives, cardiovascular drugs,antiarrhythmics, antioxicants, anti-asthma agents, hormonal agentsincluding contraceptives, sympathomimetics, diuretics, lipid regulatingagents, antiandrogenic agents, antiparasitics, anticoagulants,neoplastics, antineoplastics, hypoglycemics, nutritional agents andsupplements, growth supplements, antienteritis agents, vaccines,antibodies, diagnostic agents, and contrasting agents.

Specific examples of active agents suitable for covalent attachment to apolymer of the invention include aspariginase, amdoxovir (DAPD), antide,becaplermin, calcitonins, cyanovirin, denileukin diftitox,erythropoietin (EPO), EPO agonists (e.g., peptides from about 10-40amino acids in length and comprising a particular core sequence asdescribed in WO 96/40749), dornase α, erythropoiesis stimulating protein(NESP), coagulation factors such as Factor V, Factor VII, Factor VIIa,Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, vonWillebrand factor; ceredase, cerezyme, α-glucosidase, collagen,cyclosporin, alpha defensins, beta defensins, exedin-4, granulocytecolony stimulating factor (GCSF), thrombopoietin (TPO), α-1 proteinaseinhibitor, elcatonin, granulocyte macrophage colony stimulating factor(GMCSF), fibrinogen, filgrastim, growth hormones human growth hormone(hGH), growth hormone releasing hormone (GHRH), GRO-beta, GRO-betaantibody, bone morphogenic proteins such as bone morphogenic protein-2,bone morphogenic protein-6, OP-1; acidic fibroblast growth factor, basicfibroblast growth factor, CD-40 ligand, heparin, human serum albumin,low molecular weight heparin (LMWH), interferons such as interferonalpha, interferon beta, interferon gamma, interferon omega, interferontau, consensus interferon; interleukins and interleukin receptors suchas interleukin-1 receptor, interleukin-2, interleukin-2 fusion proteins,interleukin-1 receptor antagonist, interleukin-3, interleukin-4,interleukin-4 receptor, interleukin-6, interleukin-8, interleukin-12,interleukin-13 receptor, interleukin-17 receptor; lactoferrin andlactoferrin fragments, luteinizing hormone releasing hormone (LHRH),insulin, pro-insulin, insulin analogues (e.g., mono-acylated insulin asdescribed in U.S. Pat. No. 5,922,675), amylin, C-peptide, somatostatin,somatostatin analogs including octreotide, vasopressin, folliclestimulating hormone (FSH), influenza vaccine, insulin-like growth factor(IGF), insulintropin, macrophage colony stimulating factor (M-CSF),plasminogen activators such as alteplase, urokinase, reteplase,streptokinase, pamiteplase, lanoteplase, and teneteplase; nerve growthfactor (NGF), osteoprotegerin, platelet-derived growth factor, tissuegrowth factors, transforming growth factor-1, vascular endothelialgrowth factor, leukemia inhibiting factor, keratinocyte growth factor(KGF), glial growth factor (GGF), T Cell receptors, CDmolecules/antigens, tumor necrosis factor (TNF), monocytechemoattractant protein-1, endothelial growth factors, parathyroidhormone (PTH), glucagon-like peptide, somatotropin, thymosin alpha 1,thymosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9,thymosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) compounds,VLA-4 (very late antigen-4), VLA-4 inhibitors, bisphosponates,respiratory syncytial virus antibody, cystic fibrosis transmembraneregulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), and anti-CMVantibody. Exemplary monoclonal antibodies include etanercept (a dimericfusion protein consisting of the extracellular ligand-binding portion ofthe human 75 kD TNF receptor linked to the Fc portion of IgG1),abciximab, afeliomomab, basiliximab, daclizumab, infliximab, ibritumomabtiuexetan, mitumomab, muromonab-CD3, iodine 131 tositumomab conjugate,olizumab, rituximab, and trastuzumab (herceptin).

Additional agents suitable for covalent attachment to a polymer of theinvention include but are not limited to amifostine, amiodarone,aminocaproic acid, aminohippurate sodium, aminoglutethimide,aminolevulinic acid, aminosalicylic acid, amsacrine, anagrelide,anastrozole, asparaginase, anthracyclines, bexarotene, bicalutamide,bleomycin, buserelin, busulfan, cabergoline, capecitabine, carboplatin,carmustine, chlorambucin, cilastatin sodium, cisplatin, cladribine,clodronate, cyclophosphamide, cyproterone, cytarabine, camptothecins,13-cis retinoic acid, all trans retinoic acid; dacarbazine,dactinomycin, daunorubicin, deferoxamine, dexamethasone, diclofenac,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estramustine,etoposide, exemestane, fexofenadine, fludarabine, fludrocortisone,fluorouracil, fluoxymesterone, flutamide, gemcitabine, epinephrine,L-Dopa, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan,itraconazole, goserelin, letrozole, leucovorin, levamisole, lisinopril,lovothyroxine sodium, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, metaraminol bitartrate,methotrexate, metoclopramide, mexiletine, mitomycin, mitotane,mitoxantrone, naloxone, nicotine, nilutamide, octreotide, oxaliplatin,pamidronate, pentostatin, pilcamycin, porfimer, prednisone,procarbazine, prochlorperazine, ondansetron, raltitrexed, sirolimus,streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide,testosterone, tetrahydrocannabinol, thalidomide, thioguanine, thiotepa,topotecan, tretinoin, valrubicin, vinblastine, vincristine, vindesine,vinorelbine, dolasetron, granisetron; formoterol, fluticasone,leuprolide, midazolam, alprazolam, amphotericin B, podophylotoxins,nucleoside antivirals, aroyl hydrazones, sumatriptan; macrolides such aserythromycin, oleandomycin, troleandomycin, roxithromycin,clarithromycin, davercin, azithromycin, flurithromycin, dirithromycin,josamycin, spiromycin, midecamycin, leucomycin, miocamycin, rokitamycin,andazithromycin, and swinolide A; fluoroquinolones such asciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin,moxifloxicin, norfloxacin, enoxacin, grepafloxacin, gatifloxacin,lomefloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin,fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin,clinafloxacin, and sitafloxacin; aminoglycosides such as gentamicin,netilmicin, paramecin, tobramycin, amikacin, kanamycin, neomycin,streptomycin, vancomycin, teicoplanin, rampolanin, mideplanin, colistin,daptomycin, gramicidin, colistimethate; polymixins such as polymixin B,capreomycin, bacitracin, penems; penicillins includingpenicllinase-sensitive agents such as penicillin G and penicillin V;penicllinase-resistant agents such as methicillin, oxacillin,cloxacillin, dicloxacillin, floxacillin, and nafcillin; gram negativemicroorganism active agents such as ampicillin, amoxicillin, andhetacillin, cillin, and galampicillin; antipseudomonal penicillins suchas carbenicillin, ticarcillin, azlocillin, mezlocillin, andpiperacillin; cephalosporins such as cefpodoxime, cefprozil, ceftbuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforamide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefinetazole, ceftazidime, loracarbef, and moxalactam;monobactams such as aztreonam; and carbapenems such as imipenem,meropenem, pentamidine isethiouate, albuterol sulfate, lidocaine,metaproterenol sulfate, beclomethasone diprepionate, triamcinoloneacetamide, budesonide acetonide, fluticasone, ipratropium bromide,flunisolide, cromolyn sodium, and ergotamine tartrate; taxanes such aspaclitaxel; SN-38, and tyrphostines.

Preferred small molecules for coupling to a polymeric reagent of theinvention are those having at least one amino group. Preferred moleculesinclude aminohippurate sodium, amphotericin B, doxorubicin, aminocaproicacid, aminolevulinic acid, aminosalicylic acid, metaraminol bitartrate,pamidronate disodium, daunorubicin, levothyroxine sodium, lisinopril,cilastatin sodium, mexiletine, cephalexin, deferoxamine, and amifostine.

Preferred peptides or proteins for coupling to a polymeric reagent ofthe invention include EPO, IFN-alpha, IFN-beta, IFN-gamma, consensusIFN, Factor VII, Factor VIII, Factor IX, IL-2, remicade (infliximab),Rituxan (rituximab), Enbrel (etanercept), Synagis (palivizumab), Reopro(abciximab), Herceptin (trastuzimab), tPA, Cerizyme (imiglucerase),Hepatitus-B vaccine, rDNAse, alpha-1 proteinase inhibitor, GCSF, GMCSF,hGH, insulin, FSH, and PTH.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof. The above biologically activeproteins are additionally meant to encompass variants having one or moreamino acids substituted, deleted, or the like, as long as the resultingvariant protein possesses at least a certain degree of activity of theparent (native) protein.

The reactive polymeric reagents of the invention may be attached, eithercovalently or non-covalently, to a number of solid entities includingfilms, chemical separation and purification surfaces, solid supports,metal/metal oxide surfaces, such as gold, titanium, tantalum, niobium,aluminum, steel, and their oxides, and silicon oxide. Additionally, thepolymers of the invention may also be used in biochemical sensors,bioelectronic switches, and gates. The polymeric reagents of theinvention may also be employed as carriers for peptide synthesis, forthe preparation of polymer-coated surfaces and polymer grafts, toprepare polymer-ligand conjugates for affinity partitioning, to preparecross-linked or non-cross-linked hydrogels, and to preparepolymer-cofactor adducts for bioreactors.

VI. Pharmaceutical Compositions and Administration Methods

The invention also includes pharmaceutical preparations comprising aconjugate as provided herein in combination with a pharmaceuticalexcipient. Generally, the conjugate itself will be in a solid form(e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form. Thepharmaceutical preparations encompass all types of formulations and inparticular those that are suited for injection, e.g., powders that canbe reconstituted as well as suspensions and solutions.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The amount of the conjugate (i.e., the conjugate formed between theactive agent and the polymer described herein) in the composition willvary depending on a number of factors, but will optimally be atherapeutically effective dose when the composition is stored in a unitdose container (e.g., a vial). In addition, the pharmaceuticalpreparation can be housed in a syringe. A therapeutically effective dosecan be determined experimentally by repeated administration ofincreasing amounts of the conjugate in order to determine which amountproduces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects. Generally, however, the excipient will bepresent in the composition in an amount of about 1% to about 99% byweight, preferably from about 5%-98% by weight, more preferably fromabout 15-95% by weight of the excipient, with concentrations less than30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”, 19thed., Williams & Williams, (1995), the “Physician's Desk Reference”, 52nded., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3rd Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The conjugates can be administered injected parenterallyby intravenous injection, or less preferably by intramuscular or bysubcutaneous injection. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The pharmaceutical preparation can also take other forms such as syrups,creams, ointments, tablets, powders, and the like. Other modes ofadministration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

The invention also provides methods for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugated agent. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The actual dose to be administered will vary depend uponthe age, weight, and general condition of the subject, as well as theseverity of the condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 100mg, preferably in doses from 0.01 mg/day to 75 mg/day, and morepreferably in doses from 0.10 mg/day to 50 mg/day.

Optimally, cleavage of the water-soluble polymer portion, which may bedesirable to facilitate clearance from the body, can be facilitatedthrough the incorporation of one or more physiologically cleavableand/or enzymatically degradable linkages such as urethane, amide,carbonate or ester-containing linkages, as described above, into thepolymer component. In this way, clearance of the conjugate (via cleavageof individual water-soluble polymer portions) can be modulated byselecting the polymer molecular size and the type of linkage that wouldprovide the desired clearance properties. One of ordinary skill in theart can determine the proper molecular size of the polymer as well asthe cleavable functional group. Clearance properties can be evaluated bypreparing a series of polymer derivatives with different polymer weightsand cleavable functional groups, and then obtaining clearance profiles(e.g., through periodic blood or urine sampling) by administering thepolymer derivatives to a patient and taking periodic blood and/or urinesampling.

EXPERIMENTAL Example 1: Preparation of mPEG₅₀₀₀-6-O-D-galactopyranose(1)

Methoxy-polyethylene glycol (MW 5,000 Daltons) (M-PEG-OH, 5 kD, 8 g, 1.6mmol) in 50 mL of anhydrous toluene was azeotropically distilled underreduced pressure at 60° C. on a rotary evaporator. The azeotropicdistillation was repeated with 50 mL of anhydrous toluene and evaporatedto approximately 25 mL. To the solution was added 10 mL of anhydrousmethylene chloride and anhydrous triethylamine (0.39 mL, 2.8 mmol). Thesolution was stirred at room temperature under argon for 5 minutes and0.179 mL of dry methanesulfonyl chloride (2.3 mmol) was added dropwise.The solution was stirred at room temperature under argon overnight. Themixture was evaporated under vacuum to remove methylene chloride andfiltered over a bed of Celite to remove salts. The Celite was washedwith additional toluene and the combined filtrate was evaporated undervacuum. The solids were dissolved in approximately 15 mL of methylenechloride and precipitated on ice by the addition of isopropanol. Theprecipitated product was filtered off, washed with diethylether anddried under reduced pressure. Yield 7.7 g

¹H-NMR (d₆-DMSO, 400 MHz): δ (ppm) 3.18 (s, 3H, —SO₂—CH₃); 3.24 (s, 3H,—OCH₃); 3.51 (s, PEG backbone); 4.31 (—CH₂-OMs).

M-PEG-OMs (5 kD, 3 g, 0.6 mmol), prepared as described above, in 20 mLof anhydrous toluene was azeotropically distilled under reduced pressureat 60° C. on a rotary evaporator. The azeotropic distillation wasrepeated with 30 mL of anhydrous toluene and evaporated to approximately10 mL. To the solution was added 0.47 mL of1,2:3,4-di-O-isopropylidene-D-galactopyranose (2.0 mmol) and 0.77 g ofsodium hydride, 60% dispersion in mineral oil (19.3 mmol). The reactionwas heated at 75° C. with stirring overnight under an argon atmosphere.The reaction mixture was cooled to room temperature, diluted withmethylene chloride and filtered over a bed of Celite. The Celite waswashed with additional methylene chloride and the combined filtrate wasevaporated under vacuum. The solids were dissolved in 18 mL of 1:1methylene chloride/toluene and precipitated by the addition of 100 mLisopropanol and 40 mL diethylether at 0° C. The precipitated product wasfiltered off, washed with diethylether and dried under reduced pressure.Yield 3.15 g

¹H-NMR (CDCl₃, 400 MHz): δ (ppm) 1.21 (d, 6H, (O)₂C(CH₃)₂); 1.44 (s,3H), 1.53 (s, 3H) ((O)₂C(CH₃)₂); 3.38 (s, 3H, —OCH₃); 3.64 (s, PEGbackbone); 3.81 (t, 1H); 4.26 (m, 1H); 4.30 (m, 1H); 4.58 (m, 1H); 5.53(d, 1H).

Deprotection: The product above (2 g, 0.36 mmol) was dissolved at 0° C.in 10 mL of 8:2 trifluoroacetic acid/water and stirred 1 hour under anargon atmosphere. The reaction was stopped by the addition of 10 mLphosphate buffer (0.2 M, pH 9.1) and then neutralized to pH 6.8 with 3.0M sodium hydroxide. The solution was diluted with an equal amount ofhalf saturated sodium chloride and extracted 2 times with methylenechloride (200 mL). The combined organic layers were dried over anhydroussodium sulfate/anhydrous magnesium sulfate, filtered and evaporatedunder reduced pressure. The solids were dissolved in 5 mL methylenechloride and precipitated with 65 mL isopropanol and 20 mL diethyletherat 0° C. The precipitated product was filtered off, washed withdiethylether containing 0.3% 2,6-di-tert-butyl-4-methyl-phenol and driedunder vacuum. Yield 1.85 g.

¹H-NMR (d₆-DMSO+D₂O, 400 MHz): δ (ppm) 3.24 (s, 3H, —OCH₃); 3.51 (s, PEGbackbone); 3.94 (t); 4.23 (d); 4.92 (d).

Example 2: Preparation of mPEG₅₀₀₀-3-O-D-Glucose (2)

mPEG-OMs (5 kD, 7.7 g, 1.5 mmol), prepared as described in Example 1, in50 mL of anhydrous toluene was azeotropically distilled under reducedpressure at 60° C. on a rotary evaporator. The azeotropic distillationwas repeated with 60 mL of anhydrous toluene and evaporated toapproximately 50 mL. To the solution was added 1.35 g ofdiacetone-D-glucose (5.2 mmol) and 0.2 g of sodium hydride 60%dispersion in mineral oil (5.0 mmol). The reaction was heated at 75° C.with stirring overnight under an argon atmosphere. The reaction mixturewas cooled to room temperature, diluted with methylene chloride andfiltered over a bed of Celite. The Celite was washed with additionalmethylene chloride and the combined filtrate was evaporated undervacuum. The solids were dissolved in 15 mL of methylene chloride andprecipitated by the addition of 95 mL isopropanol and 95 mL diethyletherat 0° C. The precipitated product was filtered off, washed withdiethylether and dried under reduced pressure. Yield 7.3 g

¹H-NMR (CDCl₃): δ (ppm) 1.31 (s, 3H), 1.35 (s, 3H), 1.42 (s, 3H) 1.49(s, 3H) ((O)₂C(CH₃)₂); 3.38 (s, 3H, —OCH₃); 3.64 (s, PEG backbone); 3.92(d, 1H); 3.98 (m, 1H); 4.07 (m, 1H); 4.11 (m, 1H); 4.30 (m, 1H); 4.57(d, 1H); 5.87 (d, 1H).

Deprotection: The product above (2.5 g, 0.36 mmol) was dissolved at 0°C. in 10 mL of 8:2 trifluoroacetic acid/water and stirred 1 hour underan argon atmosphere. The reaction was stopped by the addition of 10 mLphosphate buffer (0.2 M, pH 9.1) and then neutralized to pH 6.8 with 3.0M sodium hydroxide. The solution was diluted with an equal amount ofhalf saturated sodium chloride and extracted 2 times with methylenechloride (200 mL). The combined organic layers were dried over anhydroussodium sulfate/anhydrous magnesium sulfate, filtered and evaporatedunder reduced pressure. The solids were dissolved in 5 mL methylenechloride and precipitated with 65 mL isopropanol and 20 mL diethyletherat 0° C. The precipitated product was filtered off, washed withdiethylether containing 0.3% 2,6-di-tert-butyl-4-methyl-phenol and driedunder vacuum. Yield 1.85 g

¹H-NMR (d₆-DMSO+D₂O, 400 MHz): δ (ppm) 2.9 to 3.2 (m); 3.24 (s, 3H,—OCH₃); 3.51 (s, PEG backbone); 4.29 (d); 4.89 (d).

Example 3. Preparation and Conjugation of a “Self Catalyzing” Reagent(3) A. Preparation of 2-hydroxy-3-amino-4-(2-imidazoyl)-butanal

The following synthetic scheme is employed to prepare the title compound(shown in protected form):

This compound is reacted with mPEG-SPA (the N-hydroxy succinimidyl esterof methoxypoly(ethylene glycol) propionic acid) to provide theamide-linked PEGylated reagent 3 (second compound in the scheme below).

This reagent can be reacted with a protein, according to proceduresdescribed above, to give the stable keto-amine conjugate shown.

Example 4: Preparation of 5-mPEG₁₀₀₀₀-2-Hydroxypentanal (mPEG-HP-ALD 10kD)

This reagent was prepared according to the scheme below.

a) allyl bromide, In (powder), ethanol, 40° C.; b) benzyl bromide, NaH,DMF; c) 9-BBN, DCM, H₂O₂/NaOH; d) mPEG-OMs 10K, NaH, toluene, 50° C.; e)sodium phosphate, Pd/C, H₂; f) 10% phosphoric acid

(a). Preparation of 2-hydroxy-1,1-dimethoxypent-4-ene (see D. Crestia etal., Tetrahedron: Asymmetry 12:869-876, 2001)

Indium powder (0.17 mol, 0.75 eq., 20 g, 100 mesh) was added to anethanol (200 mL) solution containing 2,2-dimethoxyacetaldehyde (0.23mol, 1.0 eq., 35 mL, 60 wt % solution in water) and allyl bromide (0.28mol, 1.2 eq., 24 mL). The suspension was stirred at 40° C. for 3 h. Theslurry was then centrifuged and the supernatant was decanted. Theresidual pellet was suspended in and washed 3 times with ethanol (3×150mL). The combined supernatants were filtered and evaporated at reducedpressure to provide a thick oil (45° C., clear yellow oil, 56 g). Thecrude product resulting (25 g) was extracted with brine and ethylacetate (2×). The combined organic layers were dried over sodiumsulfate, filtered and evaporated at reduced pressure. Yield was 11 g(89%) of semi-purified oil. The product was purified by silicachromatography with a hexanes/ethyl acetate gradient on a Biotage SP-4(FLASH 40+M) purification system. Purified yield 5.2 g (42%) clearcolorless oil.

¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 5.9 (m, 1H, CH₂═CH—); 5.2 (m, 2H,CH₂═CH—); 4.2 (d, 1H, —CH(OCH₃)₂); 3.7 (m, 1H, —CH(OH)—); 3.4 (ds, 6H,—OCH₃); 2.3 (dm, 2H, —CH₂—).

(b). Preparation of 2-hydroxybenzyl ester-1,1-dimethoxypentan-5-ol

In a dry flask 2-hydroxy-1,1-dimethoxypent-4-ene (2.92 g, 20 mmol) wasdissolved in anhydrous DMF (40 mL). Sodium hydride (1.0 g, 25 mmol, 1.25eq, 60 wt % dispersion in mineral oil) and benzyl bromide (2.73 mL, 23mmol, 1.15 eq) were added with stirring under an inert nitrogenatmosphere. After 2.5 hours, the reaction was carefully quenched by slowaddition of water. The product was extracted with brine and ethylacetate (2×). The combined organic layers were washed with brine (3×),dried over sodium sulfate, filtered and evaporated at reduced pressure.Crude yield 4.8 g (100%) clear oil.

¹H-NMR (CD₂Cl₂, 300 MHz): δ (ppm) 7.3 (bm, 5H, Ar); 5.9 (m, 1H,CH₂═CH—); 5.1 (m, 2H, CH₂═CH—); 4.6 (dd, 2H, —CH₂—Ar); 4.2 (d, 1H,—CH(OCH₃)₂); 3.5 (m, 1H, —CH(OBz)-); 3.4 (ds, 6H, —OCH₃); 2.3 (dm, 2H,—CH₂—).

(c). The crude 2-hydroxybenzyl ester-1,1-dimethoxypent-4-ene (4.2 g,17.8 mmol, 1.0 eq) was dissolved in dichloromethane (40 mL) containing9-BBN (0.5M in THF, 82 mL, 41 mmol, 2.3 eq) under an inert nitrogenatmosphere. After 2 hours, a mixture of 30% hydrogen peroxide solution(25 mL) and 15% sodium hydroxide (25 mL) was added by slow dropwiseaddition. (Note: Caution gas and heat evolution; add reagent mixtureslowly and carefully.) The reaction was stirred for an additional 3hours and then extracted with brine and ethyl acetate (2×150 mL). Thecombined organic layers were washed with brine (3×), dried over sodiumsulfate, filtered and evaporated at reduced pressure. Crude yield was7.0 g (155%) of clear oil. The crude product was dissolved in methanol(70 mL)/water (3.5 mL) and washed with hexanes (3×70 mL). The combinedhexane layers were back extracted with methanol (20 mL)/water (1 mL).The methanol layers were combined and extracted with brine and ethylacetate (2×150 mL). The combined organic layers were washed with brine(2×) and dried over sodium sulfate, filtered and evaporated at reducedpressure. Yield 4.6 g (100%) of clear semi-purified oil. Thesemi-purified product was purified by silica chromatography with ahexanes/ethyl acetate gradient on a Biotage SP-4 (FLASH 40+M)purification system. Purified yield 2.5 g (55%) clear colorless oil.

¹H-NMR (CD₂Cl₂, 300 MHz): δ (ppm) 7.3 (bm, 5H, Ar); 4.6 (dd, 2H,—CH₂—Ar); 4.2 (d, 1H, —CH(OCH₃)₂); 3.6 (t, 2H, —CH₂—OH); 3.4 (ds, 6H,—OCH₃; m, 1H, —CH(OBz)-); 1.7 (m, 2H, —CH₂—); 1.6 (m, 2H, —CH₂—).

(d). A mixture of mPEG₁₀₀₀₀-OMs (10 g, 1 mmol) and 2-hydroxybenzylester-1,1-dimethoxypentan-5-ol (0.51 g, 2 mmol, 2 eq) was prepared in 30mL of anhydrous toluene and azeotropically distilled under reducedpressure at 50° C. on a rotary evaporator. The mixture was evaporated todryness and then suspended in anhydrous toluene (30 mL) under an inertnitrogen atmosphere. Sodium hydride (0.11 g, 2.8 mmol, 2.8 eq, 60 wt %dispersion in mineral oil) was added and the reaction was heated to 75°C. The reaction was stirred at 75° C. for 24 hours then evaporated atreduced pressure. The thick oil was dissolved in dichloromethane (5 mL)and precipitated with the addition of isopropanol (300 mL). Theprecipitated product was filtered off, washed with isopropanol (75 mL),washed with diethylether (75 mL) and dried under reduced pressure. Yieldwas 9.8 g of an off-white powder.

¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 7.3 (bm, 5H, Ar); 4.6 (dd, 2H,—CH₂—Ar); 4.2 (d, 1H, —CH(OCH₃)₂); 3.6 (s, PEG backbone); 3.3 (s, 3H,—OCH₃); 1.6 (bm, 4H, —CH₂—CH₂—).

(e). In a Parr hydrogenation bottle 5-mPEG₁₀₀₀₀-2-hydroxybenzylester-1,1-dimethoxypentane (2.5 g) was dissolved in sodium phosphatebuffer (50 mM, pH 7.0, 50 mL). After sparging the solution withnitrogen, a 20% Pd/C paste (50.38% water, Pearlman's type, 0.5 g) wasadded to the bottle. The reaction mixture was hydrogenated (hydrogen gasat 20 psi) with stirring on a Parr apparatus. After 3 h hydrogen wasremoved from the reaction system by vacuum and the bottle wasdisconnected under a nitrogen flush. The suspension was filtered, rinsedwith water and extracted with brine and dichloromethane (2×). Theorganic layers were combined, dried over anhydrous sodium sulfate,filtered and evaporated at reduced pressure to provide a viscous oil.The product was precipitated by the slow addition of diethyl ether.Yield 2.0 g off-white powder.

¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 4.1 (d, 1H, —CH(OCH₃)₂); 3.6 (s, PEGbackbone); 3.3 (s, 3H, —OCH₃); 1.7, 1.4, 1.2, 0.8 (m, 4H, —CH₂—CH₂—).

(f). 5-mPEG_(10,000)-2-hydroxy-1,1-dimethoxypentane (1.6 g) wasdissolved in 10% phosphoric acid (25 mL) and the solution was spargedwith nitrogen. The hydrolysis reaction was stirred for 7 h and thenadjusted to pH 6.9 with sodium hydroxide (1 M). The product wasextracted with brine and dichloromethane (3×75 mL). The organic layerswere combined, dried over anhydrous sodium sulfate, filtered andevaporated in vacuo to provide a viscous oil. The PEG material wasdissolved in dichloromethane/isopropanol (1 mL:12 mL; containingbutylated hydroxytoluene (BHT), 24 mg) and precipitated by addition ofdiethyl ether (70 mL). The filter cake was washed with diethyl ether (20mL; containing BHT, 6 mg) and evaporated to dryness at reduced pressure.Yield 1.43 g off-white powder.

¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 3.6 (s, PEG backbone); 3.3 (s, 3H,—OCH₃); 1.7, 1.4, 1.2 (m, 4H, —CH₂—CH₂—).

Example 5: Preparation of 5-(mPEG_(10K)-methyl-amino)-2-hydroxypentanal(mPEG-MAHP-ALD 10 kD) (5)

This “self catalyzing” reagent was prepared according to the schemebelow.

a) TEA, MsCl, toluene; b) ethanol, methyl amine 40% in water; c)mPEG-OMs 10K, NaOH, water, THF, 50° C.; d) sodium phosphate, Pd/C, H₂;e) 10% phosphoric acid

(a). Preparation of 5-(methylamino)-2-hydroxybenzylester-1,1-dimethoxypentane

In a dry flask 2-hydroxybenzyl ester-1,1-dimethoxypentan-5-ol (0.62 g,2.4 mmol) was dissolved in anhydrous toluene (25 mL). Triethylamine (0.4mL, 2.9 mmol) and methane sulfonyl chloride (0.2 mL, 2.55 mmol) wereadded to the reaction at room temperature. The reaction was stirredunder inert atmosphere for 2.5 h. The slurry was filtered (0.45 micronsyringe filter; rinsed with toluene) and the solvent was evaporated atreduced pressure. Yield 0.89 g crude oil.

¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 7.3 (bm, 5H, Ar); 4.6 (dd, 2H,—CH₂—Ar); 4.3 (d, 1H, —CH(OCH₃)₂); 4.2 (t, 2H, —CH₂-OMs); 3.4 (ds, 6H,—OCH₃; m, 1H, —CH(OBz)-); 3.0 (s, 3H, —OMs); 1.9-1.6 (m, 4H, —CH₂—CH₂—).

(b). The crude oil 5-[methanesulfonyl oxy-ester]-2-hydroxybenzylester-1,1-dimethoxypentane (0.89 g) was dissolved in anhydrous ethanol(4 mL). Methyl amine (40% solution in water, 25 mL) was added and thereaction mixture was stirred for 20 h. The reaction was extracted withbrine and dichloromethane (2×). The combined organic layers were driedover anhydrous sodium sulfate, filtered and evaporated at reducedpressure. Yield 0.65 g oil.

¹H-NMR (CDCl₃, 300 MHz): δ (ppm) 7.3 (bm, 5H, Ar); 4.6 (dd, 2H,—CH₂—Ar); 4.2 (d, 1 H, —CH(OCH₃)₂); 3.4 (ds, 6H, —OCH₃; m, 1H,—CH(OBz)-); 2.6 (t, 2H, —N(CH₃)—CH₂—); 2.4 (s, 3H, —N(CH₃)—); 1.7-1.5(m, 4H, —CH₂—CH₂—).

(c). In a flask equipped with a reflux condenser,5-[methyl-amino]-2-hydroxybenzyl ester-1,1-dimethoxypentane (0.65 g, 2.4mmol) was dissolved in tetrahydrofuran (7 mL) and deionized water (5mL). M-PEG-OMs (10 kD, 3.5 g, 0.35 mmol) and sodium hydroxide were addedwith stirring. The temperature was held at 50° C. for 16 h thenadditional tetrahydrofuran (7 mL) and deionized water (3.5 mL) wereadded. The reaction was continued at 50° C. for 24 h and then refluxedin an 85° C. bath for 4 h. The product was extracted with brine anddichloromethane (3×). The organic layers were combined, dried overanhydrous sodium sulfate, filtered and evaporated to a thick oil atreduced pressure. The product was precipitated by the slow addition ofdiethyl ether. Yield 2.94 g off-white powder.

¹H-NMR (D₂O, 300 MHz): δ (ppm) 7.3 (bm, 5H, Ar); 4.6 (dd, 2H, —CH₂—Ar);4.3 (d, 1H, —CH(OCH₃)₂); 3.6 (bs, PEG backbone); 3.3 (s, 3H, —OCH₃); 2.6(m, 2H, —CH₂—N(CH₃)—); 2.3 (m, 2H, —N(CH₃)—CH₂—); 2.1 (s, 3H,—NH(CH₃)—); 1.5-1.3 (m, 4H, —CH₂—CH₂—).

(d). In a Parr hydrogenation bottle5-[mPEG₁₀₀₀₀-methyl-amino]-2-hydroxybenzyl ester-1,1-dimethoxypentane(2.9 g) was dissolved in sodium phosphate buffer (50 mM, pH 7.0, 74 mL).The solution was sparged with nitrogen and 20% Pd/C paste (50.4% water,Pearlman's type, 0.59 g) was added to the bottle. The reaction mixturewas hydrogenated (hydrogen gas at 20 psi) with stirring on a Parrapparatus. After 30 h hydrogen was removed from reaction system byvacuum and the bottle was disconnected under a nitrogen flush. Thesuspension was filtered, rinsed with water and extracted with brine anddichloromethane (3×). The organic layers were combined, dried overanhydrous sodium sulfate, filtered and evaporated to a thick oil atreduced pressure. The product was precipitated by the slow addition ofdiethyl ether. Yield 2.3 g off-white powder.

The crude PEG product (1.4 g) was purified by ion exchangechromatography on POROS media (0.2 L, Boehringer-Mannheim, BmbH,Mannheim Germany). The amine PEG product was bound to the column andthen eluted with sodium chloride (5% solution). The desired productfractions were extracted with phosphoric acid, brine and dichloromethane(3×). The organic layers were combined, dried over anhydrous sodiumsulfate, filtered and evaporated to a viscous oil at reduced pressure.The product was precipitated by the slow addition of diethyl ether.Yield 0.81 g of an off-white powder.

¹H-NMR (D₂O, 300 MHz): δ (ppm) 4.3 (d, 1H, —CH(OCH₃)₂); 3.6 (bs, PEGbackbone); 3.3 (s, 3H, —OCH₃); 3.2 (m, 2H, —N(CH₃)—CH₂—); 2.9 (s, 3H,—NH(CH₃)—); 1.9-1.5 (m, 4H, —CH₂—CH₂—).

(e). 5-[mPEG₁₀₀₀₀-methyl-amino]-2-hydroxy-1,1-dimethoxypentane (0.7 g)was dissolved in 10% phosphoric acid (15 mL) and the solution wassparged with nitrogen. The hydrolysis reaction was stirred for 28 h andthen adjusted to pH 4.5 with sodium hydroxide (1 M). The product wasextracted with brine and dichloromethane (3×75 mL). The organic layerswere combined, dried over anhydrous sodium sulfate, filtered andevaporated to a viscous oil at reduced pressure. The oil yielded anamorphous solid after slow addition with stirring of diethyl ether (50mL). This suspension was evaporated to dryness at reduced pressure.Yield 0.54 g of an off-white powder.

¹H-NMR (D₂O, 300 MHz): δ (ppm) 3.6 (bs, PEG backbone); 3.3 (s, 3H,—OCH₃); 3.2 (bm, 2H, —N(CH₃)—CH₂—); 2.9 (s, 3H, —NH(CH₃)—); 1.9-1.5 (m,4H, —CH₂—CH₂—).

Example 6. Conjugation of Lysozyme with mPEG Reagents (a) ConjugationUsing mPEG Reagents with Aliphatic Aldehyde (Butyraldehyde) andα-Hydroxy Aldehyde (HP-ALD Reagent 4)

Sodium phosphate buffers (0.2 M) were prepared at pH 6.5, ph 7.5 and pH9.0. A lysozyme (Sigma Aldrich L-6876, EC 3.2.1.17) stock solution (10mg/mL lysozyme) was also prepared in sodium phosphate buffer (20 mM, pH7.5). The reactions in FIG. 1 were run by dissolution of the indicatedPEG product (36 mg) in a mixture of lysozyme stock solution (0.1 mL) atthe indicated pH by use of sodium phosphate buffer (0.2 M, 0.9 mL).Reactions labeled as including NaCNBH₃ additionally contained freshlyprepared sodium cyanoborohydride stock solution (25 μL; 9 mg NaCNBH₃ in1 mL sodium phosphate 0.2 M, pH 7.5). The reactions were mixed andincubated at r.t. for 18 h. Aliquots (3 μL) were taken and diluted withwater and sample buffer (NuPAGE® LDS 4× buffer, Invitrogen, 6 μL).

The samples were loaded on a SDS-PAGE gel (Invitrogen NuPAGE® 4-12%Bis-Tris, running buffer MES 1×) and run at 200 V for 45 min. The gelswere removed, rinsed, stained with SimplyBlue™ SafeStain (Invitrogen)for 1 h and then destained for more than 2 h. Results are shown in FIG.1 , with lanes as indicated below:

1) Benchmark ladder,

2) lysozyme (Lyz),

3) Lyz+mPEG₁₀₀₀₀-HP-ALD (4), pH 6.5,

4) Lyz+mPEG₁₀₀₀₀-HP-ALD (4), pH 7.5,

5) Lyz+mPEG₁₀₀₀₀-HP-ALD (4), pH 9.0,

6) Lyz+mPEG₁₀₀₀₀-HP-ALD (4), pH 7.5+NaCNBH₃,

7) Lyz+mPEG₂₀₀₀₀-butyrALD (20 kD), pH 7.5,

8) Lyz+mPEG₂₀₀₀₀-butyrALD (20 kD), pH 7.5+NaCNBH₃.

As can be seen from the gel (FIG. 1 ), conversion of the protein (lowmigration band) to the conjugate (band around the midpoint of the gel)increased as the pH of the reaction was increased (lanes 3-5), in thereaction with the α-hydroxy aldehyde (HP-ALD) reagent.

No conjugation product was detectable in the reaction of protein andPEG-butyraldehyde alone (lane 7), since the expected imine product isvery sensitive to hydrolysis. A detectable product, the reduced amine,did form in the presence of a reducing agent (NaCNBH₃) (lane 8).

(b). Conjugation Using mPEG Reagents with α-Hydroxy Acetal (HP-ALD 4Acetal) and “Self-Catalyzing” (MAHP) Reagent (5)

The reactions in FIG. 2 were conducted in a similar manner with theindicated mPEG reagent (10 mg) in a mixture of lysozyme stock solution(0.03 mL) and sodium phosphate buffer (0.2 M, 0.27 mL). The reactionswere mixed and incubated at r.t. for 24 h. Aliquots (3.5 μL) were taken,and diluted with water and sample buffer (NuPAGE® LDS 4× buffer,Invitrogen, 6 μL).

The samples were loaded on a SDS-PAGE gel (Invitrogen NuPAGE® 4-12%Bis-Tris, running buffer MES 1×) and run at 200 V for 42 min. The gelswere removed, rinsed, stained with SimplyBlue™ SafeStain (Invitrogen)for 1 h and then destained for more than 2 h. Results are shown in FIG.2 , with lanes as indicated below:

1) Benchmark ladder,

2) lysozyme (Lyz),

3) Lyz+mPEG₁₀₀₀₀-HP (4) acetal, pH 6.5,

4) Lyz+mPEG₁₀₀₀₀-HP (4) acetal, pH 9.0,

5) Lyz+mPEG₁₀₀₀₀-MAHP (5) acetal, pH 6.5,

6) Lyz+mPEG₁₀₀₀₀-MAHP (5) acetal, pH 9.0,

7) Lyz+mPEG₁₀₀₀₀-MAHP-ALD (5), pH 6.5,

8) Lyz+mPEG₁₀₀₀₀-MAHP-ALD (5), pH 9.0,

9) Lyz+mPEG₁₀₀₀₀-MAHP (5) acetal (purified, 95% sub), pH 6.5,

10) Lyz+mPEG₁₀₀₀₀-MAHP (5) acetal (purified, 95% sub), pH 9.0

As can be seen, the mPEG₁₀₀₀₀-MAHP-ALD reagent (5) (lanes 7-8) showedthe greatest amount of conjugation of all reagents tested thus far, withincreased reaction at higher pH. (The acetal is expected to beunreactive at higher pH, but could undergo deprotection to the reactivealdehyde at the lower pH. The purification procedure noted for lane 9could have also caused some deprotection to the aldehyde.)

(c). Further Conjugation Reactions Using mPEG Reagents with AliphaticAldehyde (Butyraldehyde), α-Hydroxy Aldehyde (HP-ALD, 4) and“Self-Catalyzing” (MAHP) Reagent

The reactions in FIG. 3 were run by dissolution of the indicated PEGproduct (10-20 mg) in a mixture of lysozyme stock solution (0.03 mL) andthe indicated pH of sodium phosphate buffer (0.2 M, 0.27 mL). Reactionslabeled as including NaCNBH₃ additionally contained freshly preparedsodium cyanoborohydride stock solution (7.5 μL; 9 mg NaCNBH₃ in 1 mLsodium phosphate 0.2 M, pH 6.5 or pH 7.5 as needed). The reactions weremixed and incubated at r.t. for 18 h. Aliquots (3.5 μL) were taken, anddiluted with water and sample buffer (NuPAGE® LDS 4× buffer, Invitrogen,6 μL). The samples were loaded on a SDS-PAGE gel (Invitrogen NuPAGE®4-12% Bis-Tris, running buffer MES 1×) and run at 200 V for 45 min. Thegels were removed rinsed, stained with SimplyBlue™ SafeStain(Invitrogen) for 1 h and then destained for more than 2 h. Results areshown in FIG. 3 , with lanes as indicated below:

1) Lyz+mPEG_(10K)-HP-ALD (4) (10 kD), pH 6.5,

2) Lyz+mPEG_(10K)-HP-ALD (4) (10 kD), pH 7.5,

3) Lyz+mPEG_(10K)-MAHP-ALD (5) (purified, 95% sub), pH 6.5,

4) Lyz+mPEG_(10K)-MAHP-ALD (5) (purified, 95% sub), pH 7.5,

5) Lyz+mPEG_(10K)-MAHP (5) acetal (purified, 95% sub), pH 7.5,

6) Lyz+mPEG_(20K)-butyrALD (20 kD), pH 7.5,

7) Lyz+mPEG_(20K)-butyrALD (20 kD), pH 6.5+NaCNBH₃,

8) Lyz+mPEG_(20K)-butyrALD (20 kD), pH 7.5+NaCNBH₃,

9) lysozyme,

10) Benchmark ladder.

Again, reaction with the “self-catalyzing” reagent, mPEG-MAHP-ALD (5),at higher pH (lane 4) gave the greatest amount of conjugation. Noreaction was seen with the butyraldehyde reagent in the absence ofNaCNBH₃ (lanes 6-8). The simple α-hydroxy aldehyde reagent, mPEG-HP-ALD(4), gave a moderate amount of conjugation (lanes 1-2).

Example 7: Preparation of 5-mPEG_(20K)-2-hydroxy-pentanal (ormPEG-HP-ALD) (9)

This reagent (a higher molecular weight version of reagent 4) wasprepared according to the scheme below.

a) allyl bromide, In (powder), ethanol, 40° C.; b) 3,4-dihydro-2h-pyran, pyridinium p-toluenesulfonate, DCM; 9-BBN, DCM, H₂O₂/NaOH; c)methanesulfonyl chloride, TEA, toluene; d) mPEG-OH 20K, NaH, toluene,45° C.; e) 20% phosphoric acid.

(a) Preparation of 2-hydroxy-1,1-dimethoxy-pent-4-ene (Crestia et al.,2001)

Indium powder (0.52 mol, 1 eq., 60 g, 100 mesh) was added to an ethanol(250 mL) solution containing 2,2-dimethoxyacetaldehyde (0.52 mol, 1.0eq., 90.62 g, 60 wt % solution in water) and allyl bromide (0.79 mol,1.5 eq., 66.5 mL). The suspension was stirred at 40° C. for 3 h. Theslurry was then centrifuged and the supernatant was decanted. The pelletwas suspended and washed 3 times with ethanol (350, 350, and 450 mL).The combined supernatants were mixed with 1500 mL deionized water and1500 mL saturated sodium chloride. The crude product was extracted withchloroform (1500, 300 mL). The combined organic layers were washed with1500 mL saturated sodium chloride. The chloroform extract was dried oversodium sulfate, filtered and evaporated at reduced pressure. Yield 40.4g (53% yield) product.

¹H-NMR (CDCl₃, 500 MHz): δ (ppm) 5.9 (m, 1H, CH₂═CH—); 5.2 (m, 2H,CH₂═CH—); 4.2 (d, 1H, —CH(OCH₃)₂); 3.7 (m, 1H, —CH(OH)—); 3.4 (d, 6H,—OCH₃); 2.3 (dm, 2H, —CH₂—).

(b) Preparation of 2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-ol

In a dry flask 2-hydroxy-1,1-dimethoxy-pent-4-ene (38.84 g, 0.266 mol)was dissolved in toluene and azeotropically dried by distilling solvent(at 40° C.) until oil remained. The material was dissolved in anhydrousdichloromethane (780 mL). 3,4-dihydro-2 h-pyran (48.2 mL, 0.532 mol) andpyridinium p-toluenesulfonate (3.34 g, 0.013 mol) were added. Thereaction was monitored with thin layer chromatography (Mobile phase:60/40 ethyl acetate-hexanes, Vanillin stain). After 20 hrs,3,4-dihydro-2 h-pyran (5.0 mL, 0.06 mol) was added. The reaction wascomplete 3 hours after last addition of 3,4-dihydro-2 h-pyran. Thereaction solution was washed with saturated sodium bicarbonate solution(2×200 mL). The dichloromethane extract was dried with sodium sulfate,filtered, and evaporated at reduced pressure Crude yield: 61.67 g(100%), clear oil.

¹H-NMR (CDCl₃, 500 MHz): δ (ppm) 5.9 (m, 1H, CH₂═CH—); 5.1 (m, 2H,CH₂═CH—); 4.7, 4.9 (t, 1H, —CH); 4.2, 4.4 (dd, 1H, —CH); 4.0 (m, 1H,—CH); 3.8, 3.5 (m, 2H, —CH₂ from THP); 3.4 (d, 6H, (OCH₃)₂); 2.4 (m, 2H,—CH₂—); 1.5-1.8 (m, 6H, THP moiety).

The crude 2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-ol (58.76 g,0.255 mol) was dissolved in toluene (600 mL) and distilled until oilremained. The oil was dissolved in dichloromethane (500 mL). 9-BBN (0.5Min THF, 816 mL, 0.408 mol) was added under an inert nitrogen atmosphere.After 2 hours, a mixture of 30% hydrogen peroxide solution (500 mL) and15% sodium hydroxide (500 mL) was added by slow dropwise addition.(Note: Caution gas and heat evolution; add reagent mixture slowly andcarefully.) The reaction was stirred for an additional 4 hours andwashed reaction mixture with saturated sodium chloride (1000 mL, 400mL). The organic layer was dried over sodium sulfate, filtered andevaporated at reduced pressure. The crude product was dissolved inmethanol (1700 mL) with water (83 mL) and washed with hexanes (3×750mL). The methanol/water layer (bottom layer) was mixed with saturatedsodium chloride/deionized water/1M sodium hydroxide (1800 mL) and theproduct extracted with dichloromethane (1700 mL, 1000 mL). The organicextract was dried with sodium sulfate, filtered and evaporated atreduced pressure. Yield 80.14 g (100%) of clear semi-purified oil. Asample of the semi-purified product (5 g) was purified by silicachromatography with a methanol/dichloromethane gradient on a BiotageSP-4 (FLASH 40+M) purification system. Purified yield 3.4 g (70%) clearcolorless oil.

¹H-NMR (DMSO, 500 MHz): δ (ppm) 4.6-4.7 (t, 1H, —CH); 4.3, 3.3, (m, 2H,—CH₂); 4.2, 4.3 (d, 1H, —CH); 3.8 (m, 1H, —CH); 3.5, 3.4 (m, 2H, —CH₂);3.3 (ss, 6H, —O(CH₃)₂); 1.8-1.3 (m, 10H, —CH₂CH₂ and THP moiety).

(c) Preparation of2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate

In a dry flask 2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-ol (4g, 0.016 mol) was dissolved in toluene and azeotropically dried bydistilling solvent (at 40° C.) until oil remained. The material wasdissolved in anhydrous toluene (152 mL). Triethylamine (2.65 mL, 0.019mol) and methanesulfonyl chloride (1.33 mL, 0.017 mol) were added. After20 hours, the solution was filtered and washed with 1:1 deionizedwater/saturated sodium bicarbonate (2×400 mL). The toluene extract wasdried with sodium sulfate, filtered, and evaporated at reduced pressureCrude yield: 5.21 g (96%), clear oil.

¹H-NMR (DMSO, 500 MHz): δ (ppm) 4.6-4.7 (t, 1H, —CH); 4.2, (m, 2H, —CH₂;4.2, 4.3 (d, 1H, —CH); 3.8 (m, 1H, —CH); 3.6, 3.5 (m, 2H, —CH₂); 3.3 (s,6H, —(OCH₃)₂); 3.1 (s, 3H, —CH₃); 1.8-1.3 (m, 10H, —CH₂CH₂ and THPmoiety).

(d) Preparation of5-mPEG(20,000)-2-hydroxytetrahydropyranyl-1,1-dimethoxypentane

A mixture of mPEG-OH 20K (36.15 g, 1.8 mmol) and2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate (3.0g, 9.1 mmol) were dissolved in 360 mL of anhydrous toluene andazeotropically distilled under reduced pressure at 45° C. on a rotaryevaporator. The mixture was evaporated to dryness and then suspended inanhydrous toluene (100 mL with 100 mg BHT) under an inert nitrogenatmosphere. Sodium hydride (0.360 g, 9 mmol, 60 wt % dispersion inmineral oil) was added and the reaction was heated to 45° C. Thereaction was stirred at 45° C. for 67 hours. Additional sodium hydride(0.2 g, 8.3 mmol) was added, and the reaction was stirred at 45° C. foran additional 48 hours, then evaporated at reduced pressure. The thickoil was precipitated with the addition of isopropanol (2000 mL). Theprecipitated product was filtered, washed with isopropanol (750 mL),washed with diethyl ether (1000 mL), and dried under reduced pressure.Yield 23.1 g off-white powder (64% recovery).

¹H-NMR (CDCl₃, 500 MHz): δ (ppm) 4.8, 4.7 (t, 1, —CH); 4.4, 4.2 (d, 1H,—CH); 4.0 (m, 1H, —CH); 3.6 (s, PEG backbone); 3.3 (s, 3H, —OCH₃);1.8-1.4 (m, 8H, —CH₂ THP moeity).

(e) Preparation of 5-mPEG(20,000)-2-hydroxy-pentanal

5-mPEG(20,000)-2-hydroxytetrahydropyranyl-1,1-dimethoxypentane (23.1 g,1.2 mmol) was dissolved in 500 mL of 20% phosphoric acid solution inwater. After 20 hours, the pH of the mixture was adjusted to 6.80 with50% sodium hydroxide/water. Salts were filtered (due to saturatedphosphate salts) and the product was extracted with dichloromethane. Thedichloromethane extract was dried with sodium sulfate, filtered, andevaporated to a thick oil. Isopropanol/BHT solution was added to theoil. The precipitated material was filtered and washed withisopropanol/BHT solution. The product was washed with diethyl ether/BHTand dried under reduced pressure. Yield 18.0 g off-white powder.

¹H-NMR (CDCl₃, 500 MHz): δ (ppm) 9.7 (s, 1H, H—C═O); 4.2 (t, 1H, —CH);3.6 (s, PEG backbone); 3.3 (s, 3H, —OCH₃); 1.7-1.8 (m, 4H, —CH₂—CH₂—).

Example 8: Preparation of 5-mPEG_(20,000)-methylamino-2-hydroxy-pentanal(or mPEG-MAHP-ALD 20K) (10)

This reagent (a higher molecular weight version of reagent 5) wasprepared according to the scheme below.

a) methylamine solution; b) mPEG-OH 20K, NaOH, toluene, 75° C.; c) 1Msulfuric acid.

(a) Preparation of2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methylamine

In a dry flask2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate (8.5g, 0.016 mol) was dissolved in ethanol (40 mL). Methylamine (40% inwater, 255 mL) was added. After 17 hours at room temperature, saturatedsodium chloride solution (300 mL) was added. The product was extractedwith dichloromethane (300 mL). The dichloromethane extract was driedwith sodium sulfate, filtered, and evaporated under reduced pressure.Yield: 7.03 g (100%), clear oil.

¹H-NMR (DMSO, 500 MHz): δ (ppm) 4.6, 4.7 (t, 1H, —CH); 4.3, 4.2 (d, 1H,—CH); 3.8 (m, 1H, —CH); 3.5, 3.4 (m, 2H, —CH₂); 3.4 (d, 6H, (OCH₃)₂),2.4 (m, 2H, —CH₂), 1.8-1.3 (m, 10H, THP moiety and CH₂CH₂).

(b) Preparation of5-mPEG(20,000)-2-hydroxy-1,1-dimethoxy-pentan-5-methylamine

In a dry flask mPEG-mesylate 20K (25.0 g, 1.25 mmol) was dissolved intoluene (100 mL). Sodium hydroxide (0.5 g, 12.5 mmol) and2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methylamine (3.3 g,12.63 mmole) were added. The temperature of the reaction was 75° C.After 49 hours, diethyl ether (1000 mL) was added. The product wasfiltered and washed with diethyl ether (200 mL). The extract was driedwith sodium sulfate. The product was filtered and dried under reducedpressure. Yield: 23 g (92%), clear oil.

¹H-NMR (DMSO, 500 MHz): δ (ppm) 4.6, 4.7 (t, 1H, —CH); 4.3, 4.2 (d, 1H,—CH); 3.8 (m, 1H, —CH); 3.6 (s, PEG-backbone); 3.2 (s, 3H, OCH₃), 2.2(s, 3H, —CH₃), 1.8-1.3 (m, 10H, THP moiety and CH₂CH₂).

Crude5-mPEG(20,000)-2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methylamine(22.5 g, 1.13 mmol) was dissolved in deionized water (1500 mL). Thesolution was passed through desalting media (Purolite®). Conductivitywas 8.71 S/cm and pH:8.09. The solution was loaded onto POROS HS 50(cation exchange resin, 1300 mL). The column was washed with deionizedwater (approximately 1 column volume). Sodium chloride solution (5%) wasused to elute product from POROS HS 50 media. The solution pH wasadjusted to 2.70 with dilute sodium hydroxide solution. The product wasextracted with dichloromethane, and the dichloromethane extract wasdried with sodium sulfate, filtered and concentrated under reducedpressure overnight. Yield: 14 g (62% recovery).

¹H-NMR (D₂O, 300 MHz): δ (ppm) 4.3 (d, 1H, —CH(OCH₃)₂); 3.6 (bs, PEGbackbone); 3.3 (s, 3H, —OCH₃); 3.2 (m, 2H, —N(CH₃)—CH₂—); 2.9 (s, 3H,—NH(CH₃)—); 1.9-1.5 (m, 4H, —CH₂—CH₂—).

(c) Preparation of 5-mPEG(20,000)-methylamino-2-hydroxy-pentanal

In a dry flask5-mPEG(20,000)-2-hydroxy-1,1-dimethoxy-pentan-5-methylamine (12.63 g,0.63 mmol) was dissolved in 1M sulfuric acid (51 mL). After 20 hours,deionized water (440 mL) and 10% phosphoric acid (126 mL) were added.Sodium chloride (61 g) was added and the pH of the solution was adjustedto 2.8 with 1M sodium hydroxide. The product was extracted withdichloromethane (300, 100 mL). The organic extracts were dried withsodium sulfate and filtered. The filtrate was distilled to a thick oiland product was then precipitated with isopropanol/BHT solution. The wetcake was washed with isopropanol/BHT solution. The cake was washed withdiethyl ether/BHT solution, filtered, and dried under reduced pressure.Yield: 11.7 g (93%), clear oil.

¹H-NMR (D₂O (with trifluoroacetic acid spike), 300 MHz): δ (ppm) 3.6(bs, PEG backbone); 3.3 (s, 3H, —OCH₃); 3.2 (bm, 2H, —N(CH₃)—CH₂—); 2.9(s, 3H, —NH(CH₃)—); 1.9-1.5 (m, 4H, —CH₂—CH₂—).

Example 9: Preparation of 5-mPEG_(10,000)-piperazine-2-hydroxy-pentanal(or mPEG-Pip-HP-ALD 10K) (11)

This reagent was prepared according to the scheme below.

a) piperazine, water; b)2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate,dioxane, 1M sodium hydroxide; c) 1M sulfuric acid.

(a) Preparation of mPEG-piperazine 10K

In a round bottom flask, mPEG-mesylate 10K (31.0 g, 3.1 mmol) wasdissolved in a solution of piperazine (18.65 g, 216.5 mmol) in 95 mLdeionized water. After 48 hours, saturated sodium chloride (100 mL) anddeionized water (100 mL) were added. The product was extracted withdichloromethane (60, 60, 30 mL). The organic extracts were combined anddried with sodium sulfate, and the solution was filtered and distilledto a thick oil. The product was precipitated with 1:1isopropanol/diethyl ether containing BHT, washed withisopropanol/diethyl ether/BHT solution, and dried under vacuum. Yield:29.6 g (95% recovery).

¹H-NMR (DMSO, 500 MHz): δ (ppm) 3.6 (s, PEG backbone); 3.3 (s, 3H,—OCH₃); 2.6 (t, 4H, CH₂—CH₂); 2.4 (t, 2H, —CH₂); 2.3 (t, 4H, CH₂—CH₂).

(b) Preparation of5-mPEG(10,000)-piperazine-2-hydroxytetrahydropyranyl-1,1-dimethoxypentane

In a round bottom flask, mPEG-piperazine 10K (1.09 g, 0.11 mmol) and2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate(0.192 g, 0.59 mmol) were dissolved in 1,4-dioxane (1.3 mL) 1M sodiumhydroxide solution (1.3 mL). After 22 hours at 50° C., deionized water(10 mL) and saturated sodium chloride solution (25 mL) were added. Theproduct was extracted with dichloromethane (2×20 mL). Thedichloromethane extract was dried with sodium sulfate and filtered. Thefiltrate was distilled to a thick oil and precipitated with isopropanol.Diethyl ether and methyl-tert butyl ether were added to help withfiltration. The product was placed under vacuum to dry. Yield: 0.6 g(61% recovery).

¹H-NMR (DMSO, 500 MHz): δ (ppm) 4.6, 4.7 (t, 1H, —CH); 4.2, 4.3 (d, 1H,—CH); 3.8 (m, 1H, —CH); 3.6 (s, PEG backbone), 3.2 (s, 3H, —OCH₃); 2.42(t, 2H, —CH₂); 1.8-1.3 (m, 10H, THP moiety and —CH₂—CH₂).

(c) Preparation of 5-mPEG(10,000)-piperazine-2-hydroxy-pentanal (ormPEG-Pip-HP-ALD 10K)

In a round bottom flask,5-mPEG(10,000)-piperazine-2-hydroxytetrahydropyranyl-1,1-dimethoxypentane(0.528 g, 0.053 mmol) was dissolved in 1M sulfuric acid (2.1 mL). After19 hours at room temperature (approximately 21° C.), deionized water (20mL) and 10% phosphoric acid (5 mL) were added. Sodium chloride (2.5 g)was added and the pH was adjusted to 2.5 with 1M sodium hydroxide. Theproduct was extracted with dichloromethane. The dichloromethane extractwas dried with sodium sulfate and filtered. The filtrate was distilledto a thick oil and precipitated with a 1:1 mixture of isopropanol anddiethyl ether. The cake was washed with a 1:1 mixture of isopropanol anddiethyl ether and placed under vacuum to dry.

Yield: 0.35 g (66% recovery). ¹H-NMR (CDCl₃, 500 MHz): δ (ppm) 9.8 (s,1H, H—C═O); 3.6 (s, PEG backbone); 3.3 (s, 3H, OCH₃).

Example 10: Preparation of 5-mPEG_(20,000)-piperazine-2-hydroxy-pentanal(or mPEG-Pip-HP-ALD 20K) (12)

This reagent (a higher molecular weight version of reagent 11) wasprepared according to the scheme below.

a) piperazine, water; b)2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate,dioxane, 1M sodium hydroxide; c) 1M sulfuric acid.

(a) Preparation of mPEG-piperazine 20K

In a round bottom flask, mPEG-mesylate 20K (40.5 g, 2.0 mmol) wasdissolved in a solution of piperazine (25.2 g, 293 mmol) in 150 mLdeionized water. After 19 hours, saturated sodium chloride (125 mL) anddeionized water (125 mL) were added. The product was extracted withdichloromethane (60, 60, 30 mL), and the organic extracts were combined,washed with dilute sodium hydroxide and saturated sodium chloride (1:1,400 mL), and dried with sodium sulfate. The solution was filtered anddistilled to dryness. The product was dissolved in dichloromethane (80mL) and precipitated with isopropanol containing BHT (1400 mL, 400 mgBHT). Methyl tert butyl ether (400 mL) was added to help withfiltration. The product was washed with isopropanol/BHT solution andplaced under vacuum to dry.

Yield: 37.3 g (95% recovery).

¹H-NMR (DMSO, 500 MHz): δ (ppm) 3.6 (s, PEG backbone); 3.3 (s, 3H,—OCH₃); 2.6 (t, 4H, CH₂—CH₂); 2.4 (t, 2H, —CH₂); 2.3 (t, 4H, CH₂—CH₂).

(b) Preparation of5-mPEG(20,000)-piperazine-2-hydroxytetrahydropyranyl-1,1-dimethoxypentane

In a round bottom flask, mPEG-piperazine 20K (12.0 g, 0.6 mmol) and2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate (1.06g, 3.2 mmol) were dissolved in 1,4-dioxane (14.3 mL) and 1M sodiumhydroxide (14.3 mL). After 24 hours at 50° C.,2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate(0.521 g, 1.6 mmol) were added, and reaction was continued for anadditional 23 hours at 50° C. Deionized water (100 mL) and saturatedsodium chloride solution (100 mL) were added. The product was extractedwith dichloromethane (200, 100 mL). The dichloromethane extract wasdried with sodium sulfate and filtered. The filtrate was distilled to athick oil and precipitated with methyl-tert-butyl ether/BHT. The productwas washed with methyl-tert-butyl-ether/BHT and placed under vacuum todry. Yield: 11 g (92% recovery).

¹H-NMR (DMSO, 500 MHz): δ (ppm) 4.6, 4.7 (t, 1H, —CH); 4.2, 4.3 (d, 1H,—CH); 3.8 (m, 1H, —CH); 3.6 (s, PEG backbone), 3.2 (s, 3H, —OCH₃); 2.42(t, 2H, —CH₂); 1.8-1.3 (m, 10H, THP moiety and —CH₂—CH₂).

(c) Preparation of 5-mPEG_(20K)-piperazine-2-hydroxy-pentanal (ormPEG_(20K)-Pip-HP-ALD)

In a round bottom flask,5-mPEG(10,000)-piperazine-2-hydroxytetrahydropyranyl-1,1-dimethoxypentane(8.0 g, 0.4 mmol) was dissolved in 1M sulfuric acid (35 mL). After 18hours at room temperature (approximately 21° C.), deionized water (250mL) and 10% phosphoric acid (50 mL) were added. Sodium chloride (40 g)was added and the pH was adjusted to 2.5 with 1M sodium hydroxide. Theproduct was extracted with dichloromethane (200, 100, 100 mL). Thedichloromethane extract was dried with sodium sulfate and filtered. Thefiltrate was distilled to a thick oil and precipitated with a 1:1mixture of isopropanol and methyl tert-butyl ether with BHT. The cakewas washed with a 1:1 mixture of isopropanol and methyl tert-butyl etherwith BHT and placed under vacuum to dry.

Yield: 7.4 g (93% recovery).

¹H-NMR (CD₂Cl₂, 500 MHz): δ (ppm) 9.7 (s, 1H, H—C═O); 3.6 (s, PEGbackbone); 3.3 (s, 3H, OCH₃).

Example 11: Conjugation of Lysozyme with mPEG₂₀K-MAHP-ALD (10) andmPEG_(10K)-Pip-HP-ALD (11)

Sodium phosphate buffer (100 mM) was prepared at pH: 6.0, 6.5, and 9.1.A separate Lysozyme (Aldrich, L-6786, EC 3.2.1.17) solution was preparedusing 10 mM phosphate buffer pH:7.0 (10 mg/mL buffer). The lysozymesolution was spiked into the 100 mM phosphate buffers (pH:6.0, 6.5, and9.1) to obtain a specific concentrations to react with the PEG.

The conjugation reactions were run by dissolution of the indicated PEGproduct (10 mg, 15 mg) in the 2 mg/mL and 1 mg/mL Lysozyme solutions.The reactions were incubated at room temperature for 24 hours. Aliquotswere taken and diluted with 4× LDS buffer and deionized water to obtainsolutions that were 0.75 mg Lysozyme/mL solution. The samples wereloaded on a SDS-PAGE gel (Invitrogen NuPAGE© 4-12% BIS-TRIS, runningbuffer: MES SDS 1×). The samples were run at 200 V for 45 minutes. Thegels were removed, rinsed and stained with SimplyBlue™ SafeStain(Invitrogen) for 1 hour and destained for more than 2 hours. Results areshown in FIG. 4 , with lanes as indicated below:

1) Benchmark ladder,

2) Lysozyme (Lyz),

3) Lyz+mPEG-Pip-HP-ALD 10K (11), 25×, pH:6.5,

4) Lyz+mPEG-Pip-HP-ALD 10K (11), 50×, pH:6.5,

5) Lyz+mPEG-Pip-HP-ALD 10K (11), 50×, pH:9.1,

6) Lyz+mPEG-Pip-HP-ALD 10K (11), 25×, pH:6.0,

7) Lyz+mPEG-Pip-HP-ALD 10K (11), 50×, pH:6.0,

8) Lyz+mPEG-MAHP-ALD 20K (10), 25×, pH:6.5,

9) Lyz+mPEG-MAHP-ALD 20K (10), 50×, pH:6.5,

10) Lyz+mPEG-MAHP-ALD 20K (10), 50×, pH:9.1

The results in FIG. 4 show evidence of monoPEGylated and diPEGylatedconjugates of the protein were obtained using both reagents (withreagent 10 providing higher molecular weight conjugates, as expected).Greater amounts of conjugate were obtained at higher pH, which isconsistent with the base-promoted self catalyzing feature of thesereagents.

Example 12: Conjugation of Protein (55 kDa) with mPEG-HP-ALD 20K (9) andmPEG-MAHP-ALD 20K (10)

Conjugation to 5-mPEG(20,000)-2-hydroxy-pentanal (mPEG-HP-ALD 20K, 9)and “self catalyzing” reagent5-mPEG(20,000)-methylamino-2-hydroxy-pentanal (mPEG-HP-MALD 20K, 10) wasinvestigated further using a protein having a molecular weight of 55kDa. Conjugation reactions were set up by mixing a known amount ofprotein, typically around 1 mg/mL, to each PEG reagent at a molar excessof approx. 20. The conjugation reactions were carried out at pH rangesof 6.5-9.0 using a 0.2M phosphate buffer containing sucrose and allowedto proceed for up to 16 hrs at 25° C.

The resulting conjugates were analyzed by SDS-PAGE. Briefly, 45 μL ofeach sample conjugate was mixed with 15 μL of 4× LDS NuPAGE® samplebuffer and separated on a 4-12% BisTris NuPAGE® gel using the MES buffersystem, with lanes as indicated below:

1) MW markers,

2) 1 μg mPEG-HP-ALD 20K (9),

3) 3 μg unmodified 55 kDa protein,

4) mPEG-HP-ALD 20K (9)/55 kDa protein; pH 6.5,

5) mPEG-HP-ALD 20K (9)/55 kDa protein; pH 7.5,

6) mPEG-HP-ALD 20K (9)/55 kDa protein; pH 9.0,

7) 1 μg mPEG-HP-MALD 20K (10),

8) 3 μg unmodified 55 kDa protein,

9) mPEG-HP-MALD 20K (10)/55 kDa protein; pH 6.5,

10) mPEG-HP-MALD 20K (10)/55 kDa protein; pH 7.5,

11) mPEG-HP-MALD 20K (10)/55 kDa protein; pH 9.0,

12) Blank.

The data is shown in FIGS. 5A-B (5A: Barium/Iodine stain; 5B: Coomassiestain). The protein conjugate (for both panels) is indicated by thearrow to the right of the figure.

Example 13: Conjugation of Insulin with mPEG-MAHP-ALD 20K (10)

Conjugation of 5-mPEG_(20,000)-methylamino-2-hydroxy-pentanal(mPEG-HP-MALD 20K) was investigated using insulin. The conjugationreaction was performed with mPEG-MAHP-ALD 20K (25 mg) added into 0.5 mL,3 mg/ml insulin solution (50% ethanol/water, pH 9.7). The reactionmixture was incubated at room temperature with stirring at 600 rpm.After 18 hours, aliquots were taken and analyzed by reverse phase HPLC(0.1% TFA water/acetonitrile gradient, Agilent Zorbax C18, UV 276 nm).HPLC results demonstrated 20% and 27% of mono and di-PEGylated insulinconjugate formed, respectively.

Example 14: Conjugation of Lysozyme with mPEG-HP-ALD 20K (9),mPEG-MAHP-ALD 20K (10), mPEG-Pip-HP-ALD 10K (11) and mPEG-Butyraldehyde20K (Varied pH and Temperature)

The conjugation reactions were conducted with the indicated mPEGreagents (21 mg, 50 eq.) dissolved in sodium phosphate buffer (0.2 M,0.27 mL) followed by addition of lysozyme stock solution (0.03 mL, 10mg/mL in 0.2M phosphate buffer). The reactions were mixed, split into 2portions and incubated at either room temperature (approximately 21° C.)or 5° C. for 24 h or 48 h. Samples were frozen (−20° C.) until analyzed.Aliquots (4 μL) were taken and diluted with water and sample buffer(NuPAGE® LDS 4× buffer, Invitrogen, 6 μL). The samples were loaded on aSDS-PAGE gel (Invitrogen NuPAGE® 4-12% Bis-Tris, running buffer MES 1×)and run at 200 V for 42 min. The gels were removed, rinsed, stained withSimplyBlue™ SafeStain (Invitrogen) for at least 1 h and then destainedfor more than 2 h. Results are shown in FIGS. 6A-B (6A: reaction at r.t.for 1 day; 6B; reaction at 5° C. for 1 day), with lanes as indicatedbelow:

1) Lyz+mPEG-HP-ALD 20K (9), pH 6.5,

2) Lyz+mPEG-HP-ALD 20K (9), pH 9.0,

3) Lyz+mPEG-MAHP 20K (10), pH 6.5,

4) Lyz+mPEG-MAHP 20K (10), pH 9.0,

5) Lyz+mPEG-Pip-HP-ALD 20K (12), pH 6.5,

6) Lyz+mPEG-Pip-HP-ALD 20K (12), pH 9.0,

7) Lyz+mPEG-butryALD 20K, pH 6.5,

8) Lyz+mPEG-ButryALD 20K+NaCNBH₃, pH 6.5.

9) lysozyme (Lyz),

10) Invitrogen Benchmark™ ladder,

The reactions were repeated at pH 7.5 (0.2M sodium phosphate buffer or0.2M HEPES buffer) at r.t. for one day. The samples were analyzed by ona SDS-PAGE as described above. Results are shown in FIG. 6C, with lanesas indicated below:

1) Lyz+mPEG-HP-ALD 20K (9), 0.2 M phosphate buffer, pH 7.5,

2) Lyz+mPEG-HP-ALD 20K (9), 0.2 M HEPES buffer, pH 7.5,

3) Lyz+mPEG-MAHP 20K (10), 0.2 M phosphate buffer, pH 7.5,

4) Lyz+mPEG-MAHP 20K (10), 0.2 M HEPES buffer, pH 7.5,

5) Lyz+mPEG-Pip-HP-ALD 20K (12), 0.2 M phosphate buffer, pH 7.5,

6) Lyz+mPEG-Pip-HP-ALD 20K (12), 0.2 M HEPES buffer, pH 7.5,

7) Lyz+mPEG-butryALD 20K, 0.2 M phosphate buffer, pH 7.5,

8) Lyz+mPEG-butryALD 20K, +NaCNBH₃, 0.2 M phosphate buffer, pH 7.5,

9) lysozyme,

10) Invitrogen Benchmark™ ladder.

As can be seen from FIGS. 6A-C, the mPEG-MAHP-ALD 20K (10) andmPEG-Pip-HP-ALD 20K (12) reagents (self-catalyzing) showed the greatestamount of conjugation for the α-hydroxy aldehyde reagents tested thusfar, with increased reaction at higher pH. The gels appear to show acoelution of the excess mPEG-Pip-HP-ALD 20K reagent (12) with themono-PEG-lysozyme conjugate (lanes 5-6) which disturbed the expectedmigration for the mono-PEG product. (Compare gel of purified reactionmixture in FIG. 7 ; Example 17 below.) The di-PEG and tri-PEG lysozymeconjugates with mPEG-Pip-HP-ALD 20K (12) (higher migrating bands inlanes 5-6) demonstrated migration more similar to that observed for theother conjugates.

The reactions incubated at 5° C. (FIG. 6B) demonstrated qualitativelyless conjugation product compared to incubations at room temperature(FIG. 6A), although lysozyme conjugate was observed formPEG_(20K)-MAHP-ALD (10) at pH 9.0 (lane 4) and mPEG_(20K)-Pip-ALD (12)at pH 6.5 and pH 9.0 (lanes 5 and 6). As observed in previousexperiments, the butyraldehyde reagent showed little or no conjugationin the absence of reducing agent (lane 7).

Example 16: Conjugation of Lysozyme at pH 6.5, 7.5 or 9.0 withmPEG_(20K)-HP-ALD (9), mPEG_(20K)-MAHP-ALD (10), mPEG_(10K)-Pip-HP-ALD(11) and mPEG_(20K)-Butryaldehyde

The reactions summarized in Table 1 were conducted with the indicatedmPEG 20K reagent (42 mg, 20 eq.) dissolved, as indicated, in sodiumphosphate buffer (0.2 M, 1.35 mL, pH as indicated) followed by additionof lysozyme stock solution (0.15 mL, 10 mg/mL in the indicated buffer).The reactions were mixed and incubated at 25° C. for 20-24 h. Aliquotswere taken and analyzed by reverse phase HPLC (0.1% TFAwater/acetonitrile gradient, Agilent Zorbax 300SB C₃, UV 280 nm). HPLCresults demonstrated the following results for conjugate yields(relative area percent based on UV 280).

As observed previously, conjugation yields with the α-hydroxy reagentsdisclosed herein increased with pH, particularly for the“self-catalyzing” reagents. No conjugation was observed for thebutyraldehyde reagent in the absence of reducing agent.

TABLE 1 HPLC analysis of Lysozyme Conjugation Reactions with PEGAldehyde Reagents % Mono % Di % Tri mPEG % Lyso- Con- Con- Con- Reactionreagent pH zyme jugate jugate jugate 1 HP-ALD 6.5 90.2 7.1  1.1 — 2HP-ALD 7.5 85.6 10.5  2.4 — 3 HP-ALD 9.0 78.0 16.4  3.8  0.6 4 MAHP-ALD6.5 88.3 9.2  1.8 — 5 MAHP-ALD 7.5 77.0 15.3  5.3  0.9 6 MAHP-ALD 9.067.2 24.5  5.8  1.1 7 Pip-HP-ALD 6.5 88.1 6.2 — — 8 Pip-HP-ALD 7.5 80.512.4 ~2.4 ~1.8 9 Pip-HP-ALD 9.0 70.4 20.6 ~4.3 ~1.8 10 ButyrALD 6.5 98.7— — — 11 ButyrALD + 6.5 35.3 45.6 14.4  2.4 NaCNBH3

Example 17: Conjugation of Lysozyme at pH 6.5, 7.5 or 9.0 withmPEG_(20K)-Pip-HP-ALD (12), with Removal of Excess PEG Reagent

The reactions summarized in Table 2 were conducted with the indicatedmPEG 20K reagent (104 mg, 50 eq.) dissolved, as indicated, in sodiumphosphate buffer (0.2 M, 1.35 mL, pH as indicated) followed by additionof lysozyme stock solution (0.15 mL, 10 mg/mL in the indicated buffer).These reactions were similar to those summarized in lines 7-9 of Table1, but with a higher excess of PEG reagent (50× vs. 20×). The reactioncomponents were mixed and incubated at 25° C. for 20-24 h.

The reaction mixture was purified to remove excess PEG reagent, and theconjugates, including unreacted protein, were analyzed by reverse phaseHPLC (0.1% TFA water/acetonitrile gradient, Agilent Zorbax 300SB C₃, UV280 nm). HPLC results demonstrated the following results for conjugateyields (relative area percent based on UV 280).

TABLE 2 HPLC Analysis of Purified mPEG_(20K)-Pip-HP-ALD (50×)Conjugation with Lysozyme at pH 6.5, 7.5 and 9.0 mPEG-20K % % Mono % Di% Tri Reagent pH Lysozyme Conjugate Conjugate Conjugate Pip-HP-ALD 6.583.1 15.4 1.5 — Pip-HP-ALD 7.5 74.3 23.7 2   — Pip-HP-ALD 9.0 64.0 32.63.4 —

The purified reaction mixture was also analyzed by SDS-PAGE. Thus, theconjugate pools were mixed with sample buffer (NuPAGE LDS 4× buffer,Invitrogen, 10 μL) and loaded on a SDS-PAGE gel (Invitrogen NuPAGE®4-12% Bis-Tris, running buffer MES 1×) and run at 200 V for 35 min.Similar quantities of conjugate pool were loaded in each lane. The gelswere removed, rinsed, stained with SimplyBlue™ SafeStain (Invitrogen)for at least 1 h and then destained for more than 2 h. Results are shownin FIG. 8 , with lanes as indicated below:

Lane:

0) Invitrogen Mark12′

1) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 6.5;

2) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 7.5;

3) Lyz+mPEG_(20K)-Pip-HP-ALD (12), pH 9.0.

As shown by the results in FIG. 7 , increasing levels of mono-conjugatewere evident as the pH of the reaction was increased.

Example 18: Preparation of mPEG-5-O—3-Deoxyribose Reagent(5-mPEG_(20K)-2,4-Hydroxy-Pentanal or mPEG_(20K)-3d-HP-ALD) (13)

The following synthetic scheme is employed to prepare the titlecompound:

a) OsO₄ on polymer, acetone/H₂O, 30% H₂O₂; b) PhCHBr2, pyridine; c)mPEG-OMs 20K, toluene, NaH; d) Pd/C, HOAc, H₂; e) 20% phosphoric acid.

An alternate route is shown below. The starting material, methyl3-deoxy-□-D-ribofuranoside, is prepared as described in J. Am. Chem.Soc., 1964, 86(14), 2952.

a) acetone, p-toluenesulfonic acid; b) mPEG-OMs 20K, toluene, NaH; c)TFA/H₂O 8:1

This reagent can be reacted with a protein or other amine-containingcompound, according to procedures described above, to give a stableketo-amine conjugate.

Example 19: Preparation of 5-ruPEG2_(20K)-piperazine-2-hydroxy-pentanal(or ruPEG2_(20K)-Pip-HP-ALD) (14)

The synthetic scheme shown in FIG. 8 is employed to prepare the titlecompound. Briefly, the branched PEG reagent ruPEG2_(20K)-NHS (which iscommercially available from Shearwater Polymers, Inc., Huntsville Ala.)is reacted with piperazine in acetonitrile to provide the piperazineamide (step a). The free ring nitrogen is then reacted with2-hydroxytetrahydropyranyl-1,1-dimethoxy-pentan-5-methanesulfonate indioxane/1 M sodium hydroxide to give the protected precursor to thetitle compound (step b). Deprotection with 1 M sulfuric acid (step c)gives the title reagent (14). This reagent can be reacted with a proteinor other amine-containing compound, according to procedures describedabove, to give a stable keto-amine conjugate.

The invention claimed is:
 1. A polymeric conjugate having a structureIV:

where R¹ is selected from H, lower alkyl, and alkoxyalkyl; R⁴ is a two-or three-carbon chain which may be substituted with one or more groupsselected from alkyl, alkenyl, aryl, alkoxy, halo, cyano, and a watersoluble polymer, wherein the carbon adjacent to the carbonyl carbon isnot substituted with hydroxy, and wherein two substituents on R⁴ maytogether form an aliphatic or aromatic ring; NR⁵ is a secondary ortertiary amino group which is linked to a water soluble polymer, POLY,via an optional spacer group Z, where R⁵ is hydrogen or an alkyl groupwhich may form a ring with spacer group Z when present; and —NH—Brepresents the residue of an amine-containing biologically activecompound.
 2. The conjugate of claim 1, where POLY is a polyethyleneglycol, and R¹ is H or methyl.
 3. The conjugate of claim 2, where R¹ isH.
 4. The conjugate of claim 1, where R⁴ is unsubstituted.
 5. Theconjugate of claim 1, where R⁵ is lower alkyl.
 6. The conjugate of claim5, where R⁵ is methyl.
 7. The conjugate of claim 1, where NR⁵ togetherwith Z forms a piperazine ring to which POLY is linked via a ringnitrogen atom.
 8. The conjugate of claim 1, wherein the amine-containingbiologically active compound is a polypeptide or protein.
 9. Theconjugate of claim 1, wherein R⁴ is a three-carbon chain which may besubstituted with one or more groups selected from alkyl, alkenyl, aryl,alkoxy, halo, cyano, and a water soluble polymer, wherein the carbonadjacent to the carbonyl carbon is not substituted with hydroxy, andwherein two substituents on R⁴ may together form an aliphatic oraromatic ring.
 10. The conjugate of claim 9, having a structure:

where POLY is a polyethylene glycol, and each of the substituents R^(c),R^(c′), R^(d), and R^(d′) is independently selected from hydrogen,alkyl, alkenyl, aryl, alkoxy, halo, cyano, and a water soluble polymer,and each of the substituents R^(b) and R^(b′) is independently selectedfrom hydrogen, alkyl, alkenyl, aryl, alkoxy, halo, cyano, and a watersoluble polymer, wherein at most one of these substituents is a watersoluble polymer, and wherein any two of these substituents can togetherform an aliphatic ring.
 11. The conjugate of claim 10, wherein thesubstituents R^(b), R^(b′), R^(c), R^(c′), R^(d), and R^(d′) areindependently selected from hydrogen and alkyl, and further wherein anytwo such alkyl substituents can together form a 5- to 7-memberedaliphatic ring.
 12. The conjugate of claim 11, wherein R⁵ is methyl andR^(b), R^(b′), R^(c), R^(c′), R^(d), and R^(d′) are each hydrogen. 13.The conjugate of claim 1, wherein POLY is a poly(ethylene glycol). 14.The conjugate of claim 13, wherein POLY is linear.
 15. The conjugate ofclaim 13, wherein POLY is branched and has two polymer arms.
 16. Theconjugate of claim 13, wherein POLY has a weight average molecularweight of from about 100 daltons to about 150,000 daltons.
 17. Theconjugate of claim 16, wherein POLY has a weight average molecularweight of from greater than about 5,000 daltons to about 100,000daltons.